Sphingolipids signature in plasma and tissue as diagnostic and prognostic tools in oral squamous cell carcinoma.

Enzymes related to sphingolipids metabolism has been suggested as altered in oral squamous cell carcinoma (OSCC). However, clinical relevance of diverse sphingolipids in OSCC is not fully known. Here, we evaluated sphingolipidomics in plasma and tumor tissues as a tool for diagnosis/prognosis in OSCC patients. Plasma was obtained from 58 controls and 56 OSCC patients, and paired tumor and surgical margin tissues (n = 42). The levels of 28 sphingolipids molecules were obtained by mass spectrometry. Furthermore, sphingolipids were analyzed with clinical and pathological characteristics to search the potential for diagnosis and prognosis. Lower levels of 17 sphingolipids was found in the plasma of OSCC patients compared to controls while four were elevated in tumor tissues. C18:0 dyhidroceramide and C24:0 lactosylceramide in plasma were associated with perineural invasion, while tissue levels of ceramide and dyhidroceramide were associated with advanced tumor stage and perineural invasion. High plasma levels of C24:0 ceramide (HR = 0.10, p = 0.0036) and C24:1 glucosylceramide (HR = 6.62, p = 0.0023), and tissue levels of C24:0 dyhidroceramide (HR = 3.95, p = 0.032) were identified as independent prognostic factors. Moreover, we identified signatures composed by i) sphinganine-1-phosphate and C16 ceramide-1-phosphate in plasma with significant diagnostic accuracy, while ii) C24:0 ceramide, C24:0 dyhidroceramide, and C24:1 glucosylceramide plasma levels, and iii) C24:0 dyhidrosphingomyelin and C24:0 ceramide tissue levels showed value to predict survival in patients aged 60 years or older. We proposed the sphingolipids signatures in plasma and tumor tissues as biomarkers candidates to diagnosis and prognosis in OSCC.

Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of Oral Venglustat in Healthy Volunteers.

Venglustat is a small-molecule glucosylceramide synthase (GCS) inhibitor designed to reduce the production of glucosylceramide (GL-1) and thus is expected to substantially reduce formation of glucosylceramide-based glycosphingolipids. Because of its effect on glycosphingolipid formation, GCS inhibition has therapeutic potential across many disorders affecting glycosphingolipid metabolism. Therefore, venglustat is under development for substrate reduction therapy in multiple diseases, including Gaucher disease type 3, Parkinson's disease associated with GBA mutations, Fabry disease, GM2 gangliosidosis, and autosomal dominant polycystic kidney disease. Phase 1 studies were conducted in healthy volunteers to determine venglustat pharmacokinetics, pharmacodynamics, safety, and tolerability and to assess food effects on pharmacokinetics (single-dose and food-effect studies: NCT01674036; repeated-dose study: NCT01710826). Following a single oral dose of venglustat l-malate (2, 5, 15, 25, 50, 100, or 150 mg), venglustat demonstrated linear pharmacokinetics, rapid absorption (median tmax , 3.00-5.50 hours), systemic exposure unaffected by food, low apparent total body clearance (mean CL/F, 5.18-6.43 L/h), and pooled geometric mean t1/2z of 28.9 hours. Following repeated once-daily oral doses of venglustat l-malate (5, 10, or 20 mg) for 14 days, apparent steady state occurred within 5 days of repeated dosing, with pooled accumulation ratios of 2.10 for Cmax and 2.22 for AUC0-24 , and no statistically significant effect of dose or sex on accumulation. The mean fraction of dose excreted unchanged in urine (fe0-24 ) was 26.3% to 33.1%. Plasma GL-1 and GM3 decreased time- and dose-dependently. Venglustat demonstrated a favorable safety and tolerability profile.

A Randomized Study of the Effect of Replacing Sugar-Sweetened Soda by Reduced Fat Milk on Cardiometabolic Health in Male Adolescent Soda Drinkers.

Soda consumption in adolescents has been linked to poorer metabolic outcomes. We tested whether replacing soda with reduced fat milk would improve features of atherogenic dyslipidemia and other cardiometabolic risk factors. Thirty overweight and obese adolescent boys who were habitual consumers of sugar-sweetened beverages were randomly assigned to consume 24 oz/day of sugar-sweetened soda or an energy equivalent of reduced fat (2%) milk for 3 weeks with crossover to the alternate beverage after a ≥ 2 weeks washout. Plasma lipids and lipoproteins and other laboratory measures were assessed after each beverage period. Lipid and lipoprotein measurements, C-reactive protein, and serum transaminases did not differ significantly between the soda and milk phases of the study. Systolic blood pressure z-score and uric acid concentration were significantly lower after consuming milk compared to soda. Milk consumption also significantly decreased plasma glucosyl ceramide (d18:1/C16:0) and lactosylceramides (d18:1/C16:0 and d18:1/C18:0). While no effects of replacing soda with milk on lipid and lipoprotein measurements were observed in these normolipidemic weight-stable adolescent boys, decreases in systolic blood pressure, uric acid, and glycosphingolipids suggest that an overall favorable effect on cardiometabolic risk can be achieved following a short-term dietary intervention.

Liver involvement in Gaucher disease: A practical review for the hepatologist and the gastroenterologist.

Gaucher disease (GD), a rare lysosomal storage disorder caused by deficient glucocerebrosidase activity and consequent accumulation of glycosphingolipids in the mononuclear phagocyte system, may progress to disabling and potentially life-threatening complications when left undiagnosed and untreated. Unfortunately, because of non-specific signs and symptoms and lack of awareness, patients with type 1 GD, the most common non-neuropathic variant, frequently experience diagnostic delays. Since splenomegaly and thrombocytopenia are the dominant clinical features in many GD patients leading to first medical contact, the hepatologist and the gastroenterologist need to be aware of this condition. Liver involvement has been reported in the majority of GD patients, and comprises hepatomegaly, with or without liver enzymes alteration, fibrosis/cirrhosis, portal hypertension, focal liver lesions, and cholelithiasis. Moreover, GD is associated with several biochemical alterations of potential interest for the hepatologist and the gastroenterologist, including hypergammaglobulinemia, hyperferritinemia and metabolic abnormalities, that may lead to misdiagnoses with chronic liver diseases of common etiology, such as primary hemochromatosis, autoimmune liver diseases or nonalcoholic fatty liver disease. This comprehensive review, based on the collaborative experience of physicians managing patients with GD, provides practical information on the clinical, histological and radiological hepatic manifestations of GD aiming at facilitating the diagnosis of GD for the hepatologist and the gastroenterologist.

Ophthalmic manifestations of Gaucher disease: the most common lysosomal storage disorder.

Gaucher disease (GD) results from a deficiency of glucocerebrosidase activity and the subsequent accumulation of the enzyme's metabolites, principally glucosylsphingosine and glucosylceramide. There are three principal forms: Type I, which is the most common, is usually considered non-neuronopathic. Type II, III and IIIc manifest earlier and have neurological sequelae due to markedly reduced enzyme activity. Gaucher's can be associated with ophthalmological sequelae but these have not been systematically reviewed. We therefore performed a comprehensive literature review of all such ophthalmic abnormalities associated with the different types of Gaucher disease. We systematically searched the literature (1950 - present) for functional and structural ocular abnormalities arising in patients with Gaucher disease and found that all subtypes can be associated with ophthalmic abnormalities; these range from recently described intraocular lesions to disease involving the adnexae, peripheral nerves and brain. In summary, Gaucher can affect most parts of the eye. Rarely is it sight-threatening; some but not all manifestations are amenable to treatment, including with enzyme replacement and substrate reduction therapy. Retinal involvement is rare but patients with ocular manifestations should be monitored and treated early to reduce the risk of progression and further complications. As Gaucher disease is also associated with Parkinsons disease and may also confer an increased risk of malignancy (particularly haematological forms and melanoma), any ocular abnormalities should be fully investigated to exclude these potential underlying conditions.

Plasma glucosylceramides and cardiovascular risk in incident hemodialysis patients.

BACKGROUND: Recent population-based studies identified plasma sphingolipids as independent predictors of increased cardiovascular disease (CVD) morbidity and mortality. Understanding the impact of sphingolipids on CVD outcomes in patients on dialysis, who suffer from higher risk of these conditions, is important for risk assessment and treatment. OBJECTIVE: To measure plasma sphingolipid levels and determine their associations with CVD in adults initiating maintenance hemodialysis. METHODS: To evaluate associations of plasma sphingolipids with intermediate cardiovascular outcomes (hypertension, left ventricular hypertrophy, and decreased ejection fraction), cardiovascular mortality, and all-cause mortality in patients with end-stage renal disease, we measured plasma levels of ceramides, glucosylceramides, and lactosylceramides from the family of sphingolipids in 368 incident hemodialysis patients enrolled in the Predictors of Arrhythmic and Cardiovascular Risk in End-Stage Renal Disease study. RESULTS: Glucosylceramide C16GC (per 1 log µM increase) was associated with higher odds of having uncontrolled hypertension (odds ratio [OR]: 1.34; 95% confidential interval [CI]: 1.01-1.76), left ventricular hypertrophy (OR: 1.53; 95% CI: 1.11-2.13), and reduced ejection fraction (OR: 1.05; 95% CI: 1.00-1.11) in fully adjusted models. During a median 2.5 years of follow-up, there were 78 deaths from all causes, of which 33 were from CVD. Mortality was higher among those in the highest tertile of C16GC for all causes (HR: 1.81; 95% CI: 1.02-3.22) and CVD (HR: 2.63, 95% CI: 1.08-6.55). CONCLUSIONS: These results suggest that abnormal glycosphingolipid metabolism might contribute to increased CVD risk in end-stage renal disease.

Liver involvement in Gaucher disease - Review and clinical approach.

Gaucher disease (GD), one of the most prevalent lysosomal storage diseases, is associated with glucocerebroside accumulation in cells of the monocyte-macrophage system in various organs, including the liver. Evaluating and managing liver disease in patients with Gaucher disease may be challenging. While hepatic involvement is common in Gaucher disease, its severity, and clinical significance span a wide spectrum, ranging from sub-clinical involvement to liver cirrhosis with its associated complications including portal hypertension. Apart from liver involvement in Gaucher disease, patients with may also suffer from other comorbidities involving the liver. That Gaucher disease itself can mimic hepatic lesions, affect laboratory tests used to characterize liver disease, and may be associated with non-cirrhotic portal hypertension, complicates the diagnostic approach even more. Better understanding of liver involvement in Gaucher disease can spare patients unnecessary invasive testing, and assist physicians in decision making when evaluating patients with Gaucher disease suspected for significant liver disease. This review describes the various clinical manifestations, laboratory and imaging abnormalities that may be encountered when following patients with Gaucher disease for liver involvement. The mechanism for liver disease are discussed, as well as the possible hepato-protective effect of glucocerebroside, and the a diagnostic and treatment approaches.

Reductions in glucosylsphingosine (lyso-Gb1) in treatment-naïve and previously treated patients receiving velaglucerase alfa for type 1 Gaucher disease: Data from phase 3 clinical trials.

Gaucher disease (GD), an autosomal recessive lipid storage disorder, arises from mutations in the GBA1 (ß-glucocerebrosidase) gene, resulting in glucosylceramide accumulation in tissue macrophages. Lyso-Gb1 (glucosylsphingosine, lyso-GL1), a downstream metabolic product of glucosylceramide, has been identified as a promising biomarker for the diagnosis and monitoring of patients with GD. This retrospective, exploratory analysis of data from phase 3 clinical trials of velaglucerase alfa in patients with type 1 GD evaluated the potential of lyso-Gb1 as a specific and sensitive biomarker for GD. A total of 22 treatment-naïve patients and 21 patients previously treated with imiglucerase (switch patients) were included in the analysis. Overall, demographics between the two groups were similar. Mean lyso-Gb1 concentrations were reduced by 302.2ng/mL from baseline to week 209 in treatment-naïve patients and by 57.3ng/mL from baseline to week 161 in switch patients, corresponding to relative reductions of 82.7% and 52.0%, respectively. In both the treatment-naïve and switch groups, baseline mean lyso-Gb1 was higher for patients with at least one N370S mutation (363.9ng/mL and 90.7ng/mL, respectively) than for patients with non-N370S mutations (184.6ng/mL and 28.3ng/mL, respectively). Moderate correlations between decreasing lyso-Gb1 levels and increasing platelet counts, and with decreasing spleen volumes, were observed at some time points in the treatment-naïve group but not in the switch group. These findings support the utility of lyso-Gb1 as a sensitive and reliable biomarker for GD, and suggest that quantitation of this biomarker could serve as an indicator of disease burden and response to treatment.

A Simple, High-Throughput Method for Analysis of Ceramide, Glucosylceramide, and Ceramide Trihexoside in Dried Blood Spots by LC/MS/MS.

A unique monophasic extraction system coupled with LC/MS/MS to reduce matrix effects for sphingolipid analysis was developed. A solvent mixture of methanol, acetonitrile, and water was identified to simultaneously extract multiple sphingolipids with broad polarity range. To reduce matrix effects, the targeted sphingolipids were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The extraction solvent was used as an isocratic mobile phase in chromatographic separation to eliminate solvent exchange steps and enable high-throughput multiple lipid assay. The assay is linear for ceramide from 0.6 to 9 µg/mL with bias <15 %. The intra-assay coefficient of variation is less than 10 % for concentrations from 1.2 to 9 µg/mL, and less than 25 % for concentrations below 1.2 µg/mL. For glucosylceramide and ceramide trihexoside the linear range is 0.05-3 µg/mL with biases <10 % and <20 %, respectively. The intra-assay coefficient of variation for these analytes is less than 10 % at concentrations from 0.4 to 3 µg/mL, and less than 25 % for concentrations below 0.4 µg/mL.

A novel approach for quantitation of glucosylceramide in human dried blood spot using LC-MS/MS.

BACKGROUND: Glucosylceramide, an efficacy biomarker for Gaucher Type 1 disease, exhibits poor solubility in polar solvents and whole blood which makes it difficult to prepare a homogenous blood standard. RESULTS: We developed a novel method using standard addition approach by spiking a small volume of analyte solution on the surface of prespotted dried blood spot. The whole spots were punched out for subsequent extraction and LC-MS/MS analysis. The assay performance met all validation acceptance criteria. Glucosylceramide concentrations in 50 paired plasma and dry blood spot samples obtained from Gaucher Type 1 patients were tested and the results demonstrated the feasibility of using the DBS method for clinical biomarker monitoring. CONCLUSION: The new approach greatly improves assay precision and accuracy.

Comprehensive lipid profiling of plasma in patients with benign breast tumor and breast cancer reveals novel biomarkers.

Abnormal lipid metabolism is a common feature in most solid tumors, and occurs in early stages of the tumor progression. As benign breast tumor is different from malignant tumor of breast cancer, it is particularly important to take benign breast tumor into consideration when investigating cancer biomarkers. In this study, by using a normal-phase/reversed-phase two-dimensional liquid chromatography-mass spectrometry (NP/RP 2D LC-MS) method, we conducted comprehensive lipid profiling in human plasma obtained from six benign breast tumor patients and five breast cancer patients, as well as nine healthy controls. As a result, 512 lipid species were successfully identified. Principal component analysis allowed clear separation of the three groups. Quantitative analysis revealed that many lipid contents were similar in benign and malignant breast tumors compared with controls, and these were proposed as potential breast tumor biomarkers other than breast cancer biomarkers. Two phosphatidylinositol (PI) species, including PI (16:0/16:1) and PI (18:0/20:4), could differentiate between benign and malignant breast tumors, as well as breast cancer patients and healthy controls, indicating that they could be utilized as potential breast cancer biomarkers. In addition, PI (16:0/18:1), phosphatidylglycerol (36:3), and glucosylceramide (d18:1/15:1) were demonstrated to be potential biomarkers to evaluate the level of malignancy of breast tumor. Taken together, our results indicate the usefulness of lipid profiling in the discrimination between patients with breast cancer and non-carcinoma lesions, which might provide assistance in clinical diagnosis.

Glucosylceramide and lysophosphatidylcholines as potential blood biomarkers for drug-induced hepatic phospholipidosis.

Drug-induced phospholipidosis is one of the major concerns in drug development and clinical treatment. The present study involved the use of a nontargeting lipidomic analysis with liquid chromatography-mass spectrometry to explore noninvasive blood biomarkers for hepatic phospholipidosis from rat plasma. We used three tricyclic antidepressants (clomipramine [CPM], imipramine [IMI], and amitriptyline [AMT]) for the model of phospholipidosis in hepatocytes and ketoconazole (KC) for the model of phospholipidosis in cholangiocytes and administered treatment for 3 and 28 days each. Total plasma lipids were extracted and measured. Lipid molecules contributing to the separation of control and drug-treated rat plasma in a multivariate orthogonal partial least squares discriminant analysis were identified. Four lysophosphatidylcholines (LPCs) (16:1, 18:1, 18:2, and 20:4) and 42:1 hexosylceramide (HexCer) were identified as molecules separating control and drug-treated rats in all models of phospholipidosis in hepatocytes. In addition, 16:1, 18:2, and 20:4 LPCs and 42:1 HexCer were identified in a model of hepatic phospholipidosis in cholangiocytes, although LPCs were identified only in the case of 3-day treatment with KC. The levels of LPCs were decreased by drug-induced phospholipidosis, whereas those of 42:1 HexCer were increased. The increase in 42:1 HexCer was much higher in the case of IMI and AMT than in the case of CPM; moreover, the increase induced by IMI was dose-dependent. Structural characterization determining long-chain base and hexose delineated that 42:1 HexCer was d18:1/24:0 glucosylceramide (GluCer). In summary, our study demonstrated that d18:1/24:0 GluCer and LPCs are potential novel biomarkers for drug-induced hepatic phospholipidosis.

Eliglustat, an investigational oral therapy for Gaucher disease type 1: Phase 2 trial results after 4 years of treatment.

Eliglustat is an investigational, oral substrate reduction therapy for Gaucher disease type 1 (GD1). Nineteen treatment-naïve patients have now completed 4years of an open-label study (NCT00358150). Mean hemoglobin level and platelet count increased by 2.3±1.5g/dL (baseline: 11.3±1.5g/dL) and 95% (baseline: 68,700±21,200/mm(3)), respectively. Mean spleen and liver volumes (multiples of normal, MN) decreased by 63% (baseline: 17.3±9.5 MN) and 28% (baseline: 1.7±0.4 MN), respectively. Median chitotriosidase and CCL-18 each decreased by 82%; plasma glucosylceramide and GM3 normalized. Mean bone mineral density T-score for the lumbar spine increased by 0.8 (60%) (baseline: -1.6±1.1). Femur dark marrow, a reflection of Gaucher cell infiltration into bone marrow, was reduced or stable in 17/18 patients. There were no bone crises. Most adverse events were mild and unrelated to treatment. These results extend the safety and efficacy of eliglustat reported at 1 and 2 years to 4 years.

Reduced glucosylceramide in the mouse model of Fabry disease: correction by successful enzyme replacement therapy.

Fabry disease is an X-linked lysosomal storage disease (LSD) caused by deficient activity of α-Galactosidase A (α-Gal A). As a result, glycosphingolipids, mainly globotriaosylceramide (Gb3), progressively accumulate in body fluids and tissues. Studies aiming at the identification of secondary lipid alterations in Fabry disease may be potentially useful for the monitorization of the response to enzyme replacement therapy (ERT) and development of future therapies. The focus of this study was to evaluate if α-Gal A deficiency has an effect on two key groups of molecules of sphingolipids metabolism: glucosylceramides (GlucCers) and ceramides (Cers). Studies performed in a mouse model of Fabry disease showed reduced level of GlucCer and normal level of Cer in plasma, liver, spleen, kidney and heart. Moreover, analysis of GlucCer isoforms in Fabry knockout mice showed that GlucCer isoforms are unequally reduced in different tissues of these animals. ERT had a specific effect on the liver's GlucCer levels of Fabry knockout mice, increasing hepatic GlucCer to the levels observed in wild type mice. In contrast to Fabry knockout mice, plasma of Fabry patients had normal GlucCer and Cer but an increased GlucCer/Cer ratio. This alteration showed a positive correlation with plasma globotriaosylsphingosine (lyso-Gb3) concentration. In conclusion, this work reveals novel secondary lipid imbalances caused by α-Gal A deficiency.

[Cliniko-laboratory indicators of efficiency fermento-substitution therapy of Gaucher disease in Ukraine].

The analysis of efficiency of treatment of 17 patients with Gaucher disease (GD) in Ukraine who had received fermento-substitution therapy for 2 years and more was conducted on the basis of clinical and laboratory monitoring data. Regular infusions of recombinant glucocerebroside reduced signs of hepatosplenomegaly and pancytopenia, reduced a bone pain and a bone crisis at the majority of patients with GD I type that led to considerable improvement of health state and improvement of patients life quality. Efficiency of treatment depended on regularity of drug administration, dosage and severity level of the disease at the start of the therapy. Adult patients were not seen to have corrections of bones and neurologic disorders after the treatment that confirmed necessity of an early initiation of the treatment, before formation of irreversible changes in these organs and systems. Chitiotriodase activity in blood plasma is the most complex laboratory indicator which displays activity of pathological process in patients with GD, therefore it is necessary to use it for an estimation of treatment efficiency to correct a recombinant glucocerebroside dosage.

Quantification of glucosylceramide in plasma of Gaucher disease patients

Gaucher disease is a sphingolipidosis that leads to an accumulation of glucosylceramide. The objective of this study was to develop a methodology, based on the extraction, purification and quantification of glucosylceramide from blood plasma, for use in clinical research laboratories. Comparison of the glucosylceramide content in plasma from Gaucher disease patients, submitted to enzyme replacement therapy or otherwise, against that from normal individuals was also carried out. The glucosylceramide, separated from other glycosphingolipids by high performance thin layer chromatography (HPTLC) was chemically developed (CuSO4 / H3PO4) and the respective band confirmed by immunostaining (human anti-glucosylceramide antibody / peroxidase-conjugated secondary antibody). Chromatogram quantification by densitometry demonstrated that the glucosylceramide content in Gaucher disease patients was seventeen times higher than that in normal individuals, and seven times higher than that in patients on enzyme replacement therapy. The results obtained indicate that the methodology established can be used in complementary diagnosis and for treatment monitoring of Gaucher disease patients.

A doença de Gaucher é uma esfingolipidose caracterizada pelo acúmulo de glicosilceramida. O objetivo deste estudo foi desenvolver metodologia baseada na extração, purificação e quantificação da glicosilceramida plasmática a qual possa ser usada em laboratórios de pesquisa clínica. Após o desenvolvimento desta metodologia, foi proposto, também, comparar o conteúdo de glicosilceramida presente no plasma de pacientes com doença de Gaucher, submetidos ou não a tratamento, com aquele de indivíduos normais. A glicosilceramida, separada de outros glicoesfingolipídios por cromatografia de camada delgada de alto desempenho (HPTLC), foi revelada quimicamente (CuSO4/H3PO4) e a respectiva banda foi confirmada por imunorrevelação (anticorpo anti-glicosilceramida humana/anticorpo secundário conjudado à peroxidase). A quantificação do cromatograma por densitometria demonstrou que o conteúdo de glicosilceramida nos pacientes com doença de Gaucher era 17 vezes maior que aquele de indivíduos normais e 7 vezes maior que aquele dos pacientes com doença de Gaucher submetidos a tratamento com terapia de reposição enzimática. Os resultados obtidos demonstram que a metodologia estabelecida pode ser usada como diagnóstico complementar e como monitoração do tratamento de pacientes com doença de Gaucher.

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.

Chronic treatment with pioglitazone does not protect obese patients with diabetes mellitus type II from free fatty acid-induced insulin resistance.

CONTEXT: Thiazolidinediones increase peripheral insulin sensitivity and decrease plasma free fatty acids (FFA). However, their exact mechanism of action has not been fully elucidated. OBJECTIVE: We studied the protective effect of pioglitazone on FFA-induced insulin resistance and the effects on intramyocellular glycosphingolipids. DESIGN: We studied glucose metabolism in the basal state and during a hyperinsulinemic euglycemic clamp by using stable isotopes. Studies were performed at baseline and after 4 months of treatment with pioglitazone. Patients were then studied on a third occasion during infusion of a lipid emulsion to increase plasma FFA to pretreatment levels. All studies were combined with muscle biopsies to measure intramyocellular ceramide and glycosphingolipids. PATIENTS: Patients were obese with poorly controlled type 2 diabetes mellitus. INTERVENTION: Patients were treated with 30 mg pioglitazone once daily. MAIN OUTCOME MEASURE: The change in peripheral insulin sensitivity after treatment with pioglitazone and during the infusion of the lipid emulsion was the main outcome measure. RESULTS: Peripheral glucose uptake (Rd) increased significantly, but returned to baseline levels after increasing plasma FFA to pretreatment levels. Insulin-mediated suppression of FFA was increased significantly. Intramyocellular ceramide concentrations were higher during the hyperinsulinemic clamp after treatment with pioglitazone, but not in the basal state. The intramyocellular content of glycosphingolipids and plasma concentrations of ceramide and glycosphingolipids did not change. CONCLUSIONS: Pioglitazone increases Rd and insulin-mediated suppression of plasma FFA, but does not protect patients with type 2 diabetes mellitus from FFA-induced insulin resistance. This effect of pioglitazone is not attained via a decrease in intramyocellular concentrations of ceramide or glycosphingolipids.

Ten years' experience of enzyme infusion therapy of Norrbottnian (type 3) Gaucher disease.

AIM: To study the long-term effects of enzyme replacement therapy on the neurological signs of chronic neuronopathic Gaucher disease and to evaluate some biochemical parameters for monitoring the treatment. METHODS: Eight patients from the Norrbottnian cohort were followed during 10 y of treatment. They were followed with regular clinical observations, biochemical tests and psychometric testing. RESULTS: After the start of treatment, their general well-being improved and was stable during the follow-up. In three of the patients there were some indications of slow neurological deterioration. The mean results of psychometric testing did not decrease. Glucosylceramide and chitotriosidase levels were useful in monitoring the treatment. The chemokine CCL18 also seems to be a useful parameter for future monitoring. CONCLUSIONS: Enzyme replacement therapy seems to slow down further neurological and mental deterioration in mild chronic neuronopathic (type 3) Gaucher disease.