Quantitative evaluation of sphingomyelin and glucosylceramide using matrix-assisted laser desorption ionization time-of-flight mass spectrometry with sphingosylphosphorylcholine as an internal standard. Practical application to tissues from patients with Niemann-Pick disease types A and C, and Gaucher disease

Niemann-Pick disease types A and C, and Gaucher disease are glycolipid storage disorders characterized by the systemic deposition of glycosphingolipids, i.e., sphingomyelin in Niemann-Pick disease types A and C tissues and glucosylceramide in Gaucher disease ones, respectively. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS), we analyzed the sphingolipids in liver and spleen specimens from patients with Niemann-Pick disease types A and C, and Gaucher disease. Crude lipids were extracted from tissue containing 5mg protein with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by MALDI-TOF/MS. The results were as follows: (a) ion peaks with m/z values corresponding to different sphingomyelin and ceramide monohexoside (CMH) species were clearly detected. (b) With sphingosylphosphorylcholine as the internal standard for quantification of sphingomyelin and CMH, the relative peak heights of sphingomyelin and CMH were calculated and plotted versus their contents. The relative peak heights of sphingomyelin and CMH showed linearity between 50 and 1500 ng sphingomyelin content, and between 5 and 150 ng CMH content, respectively. (c) Quantitative analysis revealed the accumulation of sphingomyelin in the liver and spleen specimens from the patients with Niemann-Pick disease types A and C. Striking accumulation of CMH was also detected in the liver and spleen specimens from the patients with Gaucher disease. This investigation indicated that accumulated sphingomyelin and CMH in small amounts of tissues from sphingolipidosis patients can be detected quantatively with the MALDI-TOF/MS method. This method will be useful not only for the diagnosis but also for biochemical pathophysiology evaluation of patients with various sphingolipidosis.     

Evaluation of sphingolipids in vitreous bodies from a patient with Gaucher disease, using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Gaucher disease is a glycolipid storage disorder characterized by the accumulation of glucosylceramide in tissues. Using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS), we analyzed sphingolipids in vitreous bodies from a patient with Gaucher disease who suffered from vitreous opacities. Crude lipids were extracted from the freeze-dried vitreous bodies with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by DE MALDI-TOF-MS. The results were as follows: (a). the m/z values of the ions found in the mass spectra for both the control and the Gaucher disease patient corresponded to different sphingomyelin species. (b). The mass spectrum of the Gaucher disease patient showed additional ions with m/z values corresponding to different ceramide monohexoside (CMH) species. It was indicated that the accumulation of CMH in vitreous bodies from Gaucher disease patients could be easily detected with the DE MALDI-TOF-MS method.     

Sulfatide and Sphingomyelin Loading of Living Cells as Tools for the Study of Ceramide Turnover by Lysosomal Ceramidase - Implications for the Diagnosis of Farber-Disease

The ceramide turnover by lysosomal ceramidase in intact, living cells was investigated by loading radiolabeled sulfatide or sphingomyelin in situ on skin fibroblasts and lymphoid cells. The cells originated from normal individuals and from patients with acid ceramidase deficiency (Farber disease). While fibroblasts from individuals with Farber disease exhibited some impairment in the degradation of the ceramide produced by sulfatide hydrolysis, lymphoid cells from individuals with Farber disease metabolized the ceramide as readily as did normal cells, suggesting the existence in lymphoid cells of a nonlysosomal degradation pathway for the sulfatide-derived ceramide, In contrast, sphingomyelin loading in the presence of serum showed a considerably decreased turnover of ceramide in both fibroblasts and lymphoid cells from individuals with Farber disease. Further methodologic variation led to the use of LDL-associated radioactive sphingomyelin; LDL-association promoted the targeting of exogenous sphingomyelin to lysosomes. As a result, an almost complete deficiency of ceramide degradation was found in cells from severely affected patients with Farber disease. Our data with this novel method show that sphingomyelin loading of intact living cells is a simple, alternative means for determining ceramide degradation by lysosomal ceramidase and for diagnosing Farber disease.     

Niemann-Pick disease, Type C: evidence for the deficiency of an activating factor stimulating sphingomyelin and glucocerebroside degradation

1) Qualitative lipid analyses by thin-layer chromatography of 4 Niemann-Pick type C spleens confirmed sphingomyelin accumulation together with increase in the amount of glucocerebroside. 2) In the presence of crude sodium taurocholate as detergent, sphingomyelin degradation rates of normal and Niemann-Pick type C-cultured fibroblasts were fairly close under standard conditions at pH 5.0. In the absence of sodium taurocholate, sphingomyelinase activity was optimal at pH 4.0. Sphingomyelinase activities of fibroblasts from two patients with Niemann-Pick disease type C measured without detergent, were about 30% of that of controls. 3) Extracts from Gaucher spleen heated to 90 degrees C and devoid of sphingomyelinase activity stimulated at the optimal pH of 4.0 sphingomyelin degradation by cultured normal fibroblasts (2--4-fold, Niemann-Pick type C fibroblasts (5--9-fold), whereas similarly treated extracts from Niemann-Pick type C spleen showed no stimulation of sphingomyelin catabolism. Heated extracts from normal human spleen exhibited a smaller stimulation than that shown by Gaucher spleen. This stimulating effect could not be observed in fibroblasts from patients suffering from Niemann-Pick type B (sphingomyelinase defect). 4) Heat-treated extracts of Gaucher spleen were fractionated by ion exchange chromatography, isoelectric focusing and gel filtration. The active fractions obtained by these procedures stimulated sphingomyelin as well as glucocerebroside degradation and were absent from the corresponding Niemann-Pick type C preparations. Enriched activator preparations of Gaucher spleen stimulated sphingomyelinase activity of Niemann-Pick type C fibroblasts 25--38-fold and that of normal cells 3-fold. 5) The activating factor had an isoelectric point of 4.0 and an apparent molecular weight, as estimated by gel filtration, of 25000. Treatment with pronase E abolished its activity.     

An enzymatic assay for quantifying sphingomyelin in tissues and plasma from humans and mice with Niemann-Pick disease

Sphingomyelin is an important lipid component of cell membranes and lipoproteins which can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase, and the resultant accumulation of sphingomyelin in cells and tissues. In this paper we report a new, enzyme-based method to quantify the levels of sphingomyelin in tissues and plasma of normal individuals and NPD patients. The method utilizes sphingomyelinase from Bacillus cereus to completely hydrolyze the sphingomyelin into ceramide. Quantification of the sphingomyelin-derived ceramide is accomplished using Escherichia coli diacylglycerol (DAG) kinase and [gamma-(32)P]ATP. The resulting [(32)P]ceramide is quantified using a phosphor-imager system following TLC separation. This procedure allowed quantification of sphingomyelin over a broad range from 10 pmol to 1 nmol. To validate this assay we quantified sphingomyelin in plasma and tissues obtained from normal and NPD mice and humans. The sphingomyelin content in adult homozygous (-/-) or heterozygous (+/-) NPD mouse plasma was significantly elevated compared to that of normal mice (up to twofold). Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 40 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several type B NPD patients also were significantly elevated compared to normal individuals of the same age. Based on these results we propose that this new, enzyme-based procedure can provide sensitive and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could be a useful tool for the diagnosis of NPD and the evaluation of NPD treatment protocols, as well as for the study of ceramide-mediated apoptosis since the method provides the simultaneous determination of sphingomyelin and ceramide in the same lipid extract.     

A lipid analogue that inhibits sphingomyelin hydrolysis and synthesis, increases ceramide, and leads to cell death

We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 microM led to an increase in cellular ceramide and cell death. Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells.     

Uptake and intracellular degradation of fluorescent sphingomyelin by fibroblasts from normal individuals and a patient with Niemann-Pick disease

Sphingomyelin, labelled with a fluorescent probe, pyrene, in the fatty acyl residue was associated with fetal calf serum; approx. 80% of the sphingomyelin was found in the low- and high-density lipoproteins. This was added to the growth medium of cultured human skin fibroblasts from normal individuals and a patient with Niemann-Pick disease type A, devoid of acid sphingomyelinase activity. The fluorescent sphingomyelin was taken up by both cell types, but only the former degraded it to produce fluorescent ceramide. Differences between normal and Niemann-Pick cells in sphingomyelin content or ceramide production were observed after several hours uptake. A more pronounced difference was noted when cells were incubated for 1 day with fluorescent sphingomyelin and then for two to three days in medium devoid of this compound. Under these conditions, the fluorescence intensity of the Niemann-Pick cells remained practically constant while that of their normal counterparts was almost completely eliminated from the cells. Comparison of fluorescence intensities of these two cell types could be made directly on aqueous suspensions of whole cells or, alternatively, on their lipid extracts. For evaluation of the degradation of fluorescent sphingomyelin to ceramide within the cells, several procedures were developed for the rapid isolation of the latter compound from the total lipid extract. The results suggest that when associated with the constituents of the fetal calf serum, sphingomyelin is taken up by the cells and transported into the lysosomal compartment where it is degraded to ceramide. Use of the fluorescent derivative of sphingomyelin provided a simple and rapid procedure for following the uptake by and degradation within the cultured cells. It also permitted the establishment of differences in the rates of degradation of the fluorescent sphingomyelin by cells with a normal metabolism and others lacking sphingomyelinase (i.e., Niemann-Pick disease type A cells).     

Determination of 7-ketocholesterol in plasma by LC-MS for rapid diagnosis of acid SMase-deficient Niemann-Pick disease

Acid sphingomyelinase (ASMase)-deficient Niemann-Pick disease (NPD) is caused by mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene, resulting in accumulation of sphingomyelin in the lysosomes and secondary changes in cholesterol metabolism. We hypothesized that the oxidation product of cholesterol, 7-ketocholesterol (7-KC), might increase in the plasma of patients with ASMase-deficient NPD. In this study, a rapid and nonderivatized method of measurement of plasma 7-KC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Plasma samples from healthy subjects, patients with ASMase-deficient NPD, nonaffected ASMase-deficient NPD heterozygotes, Niemann-Pick type C (NPC) disease, glycogen storage disorder type II (GSDII), Gaucher disease (GD), mucopolysaccharidosis type II (MPSII), Krabbe disease (KD), and metachromatic leukodystrophy (MLD) were tested retrospectively. Markedly elevated 7-KC was found in patients with ASMase-deficient NPD and NPC disease that showed significant differences from ASMase-deficient NPD heterozygotes; patients with GSDII, GD, MPSII, KD, and MLD; and normal controls. The analysis of plasma 7-KC by LC-MS/MS offers the first simple, quantitative, and highly sensitive method for detection of ASMase-deficient NPD and could be useful in the diagnosis of both ASMase-deficient NPD and NPC disease.     

Curcumin induces apoptosis of multidrug-resistant human leukemia HL60 cells by complex pathways leading to ceramide accumulation

Most anti-cancer agents induce apoptosis, however, a development of multidrug resistance in cancer cells and defects in apoptosis contribute often to treatment failure. Here, the mechanism of curcumin-induced apoptosis was investigated in human leukemia HL60 cells and their HL60/VCR multidrug-resistant counterparts. In both cell lines curcumin induced a bi-phasic ceramide generation with a slow phase until 6 h followed by a more rapid one. The level of the ceramide accumulation correlated inversely with the cell viability. We found that the ceramide elevation resulted from multifarious changes of the activity of sphingolipid-modifying enzymes. In both cell lines curcumin induced relatively fast activation of neutral sphingomyelinase (nSMase), which peaked at 3 h, and was followed by inhibition of sphingomyelin synthase activity. In addition, in HL60/VCR cells the glucosylceramide synthase activity was diminished by curcumin. This process was probably due to curcumin-induced down-regulation of P-gp drug transporter, since cyclosporine A, a P-gp blocker, also inhibited the glucosylceramide synthase activity. Inhibition of nSMase activity with GW4869 or silencing of SMPD3 gene encoding nSMase2 reversed the curcumin-induced inhibition of sphingomyelin synthase without affecting the glucosylceramide synthase activity. The early ceramide generation by nSMase was indispensable for the later lipid accumulation, modulation of Bax, Bcl-2 and caspase 3 levels, and for reduction of cell viability in curcumin-treated cells, as all these events were inhibited by GW4869 or nSMase2 depletion. These data indicate that the early ceramide generation by nSMase2 induced by curcumin intensifies the later ceramide accumulation via inhibition of sphingomyelin synthase, and controls pro-apoptotic signaling.     

Presence of interleukin 10 in the serum and blister fluid of patients with pemphigus vulgaris and pemphigoid

Interleukin 10 (IL-10) is an immunoregulatory cytokine produced by T lymphocytes and macrophages. Recently, it has been suggested that IL-10 may be involved in the pathogenesis of various inflammatory and autoimmune diseases. Using an ELISA we investigated the presence of IL-10 in the serum and blister fluid of pemphigus vulgaris (PV) patients with active disease and those in prolonged clinical remission compared with normal controls. Sera from patients with bullous pemphigoid (BP), ocular cicatricial pemphigoid (OCP), oral pemphigoid (OP) and blister fluid from five patients with BP were also studied. Increased levels of IL-10 were detected in the sera of 87.5% of patients with active PV and were statistically significant (P=0.0003) when compared with levels in normal human serum. Lower levels of IL-10 were detected in 12.5% PV patients in remission and were statistically significant (P=0.0001) when compared with levels in patients with active disease. Levels of IL-10 were detected in sera of 4.6% (1 of 24) of the normal controls. The levels of IL-10 were approximately four times higher in blister fluids than levels in the serum in the same PV patients. This difference was highly statistically significant (P=0.0008). A correlation was observed between serum levels of IL-10 and titres of pemphigus autoantibodies and with disease severity. Elevated level of IL-10 was detected in the blister fluid from five BP patients. Levels of IL-10 in the sera of patients with BP, OCP and OP were not significantly increased. These preliminary data suggest that IL-10 in concert with other cytokines may play an important role in the pathogenesis of PV and BP.     

Splenic lipids in Gaucher's disease

Column chromatography (on cellulose, silicic acid, and Florisil) and thin-layer chromatography were employed for the separation and purification of lipid fractions from normal and Gaucher spleens. A new hydrolysis procedure, followed by paper chromatography, was used for identification of sugar moieties. A nonhydrolytic combined colorimetric procedure, with anthrone and orcinol, was used for the estimation of glucose and galactose separately in glycolipids. The limitations of this method were examined. Spleens from two control subjects and three patients with Gaucher's disease have been examined in detail. In all Gaucher spleens, the predominant feature was the massive acccumulation of glucocerebroside; neutral ceramide oligohexoside levels were probably within the normal range, as were other neutral lipids and phospholipids. In one case examined for gangliosides, these were increased twentyfold. One Gaucher spleen, in which others had reported that the stored "cerebroside" contained predominantly lactose as the saccharide moiety, has been examined in detail and it has been established that the stored material was, in fact, glucocerebroside, ceramide lactoside levels not being significantly elevated. In a further nine cases glucose was the major sugar detected in the splenic lipids.     

Brain ceramide hexosides in Tay-Sachs disease and generalized gangliosidosis (GM1-gangliosidosis)

The carbohydrate composition was determined for ceramide hexosides isolated from brains of patients with Tay-Sachs disease and generalized gangliosidosis (hereby named GM1-gangliosidosis). Gray matter of patients with each disease showed a characteristic abnormal ceramide hexoside pattern. In Tay-Sachs gray matter, ceramide trihexoside is the major component, whereas ceramide tetrahexoside is barely detectable. In GM1-gangliosidosis, ceramide tetrahexoside is the major ceramide hexoside, while ceramide trihexoside is present only in small amount. These two major components have been characterized as the asialo derivatives of, respectively, the "Tay-Sachs ganglioside" (GM2-ganglioside) and the normal major monosialoganglioside (GM1-ganglioside). In both diseases, more than half the ceramide monohexoside of gray matter was glucocerebroside. Gray matter ceramide dihexoside, present in both diseases at higher than normal levels, was mostly ceramide lactoside, with possibly a small amount of ceramide digalactoside. Sulfatide contained only galactose. The abnormal ceramide hexoside pattern is limited to gray matter: white matter showed normal ceramide hexosides, i.e. a preponderance of monohexosides and sulfatide, with no detectable glucocerebroside.     

The potential role for sphingolipids in neuropathogenesis of Alzheimer’s disease

The review discusses the functional role of sphingolipids in the pathogenesis of Alzheimer’s disease (AD). Certain evidence exists that the imbalance of sphingolipids such as sphingomyelin, ceramide, sphingosine, sphingosine-1-phosphate and galactosylceramide in the brain of animals and humans, in the cerebrospinal fluid and blood plasma of AD patients plays a crucial role in neuronal function by regulating growth, differentiation and cell death in CNS. Activation of sphingomyelinase (Smase), which leads to the accumulation of the proapoptotic agent, ceramide, can be considered as a new mechanism for AD and may be a prerequisite for the treatment of this disease by using drugs that inhibit SMase activity. The role of sphingolipids as biomarkers for the diagnosis of the early stage of Alzheimer’s disease and monitoring the effectiveness of treatment with new drugs is discussed.     

Characterization of neutral sphingolipids from chicken erythrocytes

The neutral sphingolipids from chicken erythrocytes were characterized. The total concentration of neutral sphingolipids was found to be 480 nmol/g of dry stroma. They were isolated and purified by droplet counter-current chromatography, Iatrobeads column chromatography, and preparative thin-layer chromatography. The major neutral sphingolipids were free ceramide, ceramide monohexoside, ceramide dihexoside, and ceramide pentahexoside, which represented 43%, 23.5%, 10.0%, and 3.6% of the long chain bases, respectively. Thus, free ceramide was the most abundant neutral sphingolipid in chicken erythrocytes. Ceramide monohexoside was composed of more galactosylceramide than glucosylceramide. Galabiosylceramide was found in the ceramide dihexoside fraction together with lactosylceramide. Ceramide pentahexoside was a Forssman glycolipid. There were two groups of neutral sphingolipids; one had mainly C16 fatty acid and the other had C22 and C24 fatty acids. In both groups sphingosine (d18:1) was predominant as a long chain base. 2-Hydroxy-C16 fatty acid was a major component of one of the ceramide monohexosides.     

Glycosphingolipids of cultured human colon carcinoma cells and their drug-resistant sublines

Human colon carcinoma cells were analyzed for lipid phosphorus, cholesterol and glycosphingolipids. Ceramide mono-, di- and trihexosides and sulfatides were isolated by column and thin-layer chromatography and determined quantitatively on the basis of their hexose content. The complex lipid fractions so isolated were only partially resolved with the material available. Gangliosides GM2 and GM3 and globoside were major components of the fraction and were determined on the basis of their hexose, hexosamine and neuraminic acid content. The HCT 116, 116a and 116b cells contained no fucolipids. Cell lines resistant to mitomycin C, teniposide and etoposide were developed and analyzed. Over the 5 year period of the study sulfatides declined to about one-fourth of their original amounts in both parent and drug-adapted cells. HCT 116 cells adapted to mitomycin C and teniposide had 30% less ceramide monohexoside and a 45% greater cholesterol to lipid phosphorus ratio than the parent cells. Reductions in ceramide dihexoside in the drug-adapted cells were greater than those of the ceramide monohexoside. Galabiosyl ceramide was the major ceramide dihexoside in all the cells and accumulated in HCT 116a to levels 4-6-fold greater than that of the other lines as the only dihexoside.     

AAV8-mediated expression of glucocerebrosidase ameliorates the storage pathology in the visceral organs of a mouse model of Gaucher disease

BACKGROUND: Gaucher disease is the most common of the lysosomal storage disorders. The primary manifestation is the accumulation of glucosylceramide (GL-1) in the macrophages of liver and spleen (Gaucher cells), due to a deficiency in the lysosomal hydrolase glucocerebrosidase (GC). A Gaucher mouse model (D409V/null) exhibiting reduced GC activity and accumulation of GL-1 was used to evaluate adeno-associated viral (AAV)-mediated gene therapy. METHODS: A recombinant AAV8 serotype vector bearing human GC (hGC) was administered intravenously to the mice. The levels of hGC in blood and tissues were determined, as were the effects of gene transfer on the levels of GL-1. Histopathological evaluation was performed on liver, spleen and lungs. RESULTS: Vector administration to pre-symptomatic Gaucher mice resulted in sustained hepatic secretion of hGC at levels that prevented GL-1 accumulation and the appearance of Gaucher cells in the liver, spleen and lungs. AAV administration to older mice with established disease resulted in normalization of GL-1 levels in the spleen and liver and partially reduced that in the lung. Analysis of the bronchoalveolar lavage fluid (BALF) from treated mice showed significant correction of the abnormal cellularity and cell differentials. No antibodies to the expressed hGC were detected following a challenge with recombinant enzyme suggesting the animals were tolerized to human enzyme. CONCLUSIONS: These data demonstrate the effectiveness of AAV-mediated gene therapy at preventing and correcting the biochemical and pathological abnormalities in a Gaucher mouse model, and thus support the continued consideration of this vector as an alternative approach to treating Gaucher disease.     

血清与腹腔液中趋化因子RANTES水平在子宫内膜异位症患者中的临床应用

目的:探讨血清与腹腔液中趋化因子RANTES水平在子宫内膜异位症(EM)患者中的临床意义。方法:选取2012年5月-2013年5月本院收治的33例EM患者(观察组)、33例良性卵巢肿瘤患者(对照组)和33例健康体检者(健康对照组),应用ELISA法对血清与腹腔液中趋化因子RANTES水平进行检测,分析RANTES水平与患者r-AFS分期及痛经程度的相关性。结果:观察组血清RANTES水平明显高于对照组和健康对照组,差异均有统计学意义(t=7.163,6.743,均P0.05);观察组腹腔液RANTES水平亦高于对照组,两组比较差异有统计学意义(t=5.927,P0.05);观察组血清及腹腔液中RANTES水平与r-AFS分期呈正相关(r=0.975,0.893,均P0.05),且随分期增高而呈递增趋势;观察组血清RANTES水平与患者痛经评分无明显的相关性(r=-0.312,P0.05);而腹腔液中RANTES水平与患者痛经评分呈正关(r=0.517,P0.05)。结论:EM患者血清与腹腔液中趋化因子RANTES水平明显上升,应用ELISA法检测RANTES水平可辅助EM诊断,有利于提高诊断准确率。     

Ceramide generation by two distinct pathways in tumor necrosis factor alpha-induced cell death

Ceramide accumulation in the cell can occur from either hydrolysis of sphingomyelin or by de novo synthesis. In this study, we found that blocking de novo ceramide synthesis significantly inhibits ceramide accumulation and subsequent cell death in response to tumor necrosis factor alpha. When cells were pre-treated with glutathione, a proposed cellular regulator of neutral sphingomyelinase, inhibition of ceramide accumulation at early time points was achieved with attenuation of cell death. Inhibition of both pathways achieved near-complete inhibition of ceramide accumulation and cell death indicating that both pathways of ceramide generation are stimulated. This illustrates the complexity of ceramide generation in cytokine action.     

Immunochemical characterization of two activator proteins stimulating enzymic sphingomyelin degradation in vitro. Absence of one of them in a human Gaucher disease variant

Two nonenzymic activator proteins shown previously to strongly stimulate enzymic sphingomyelin degradation in vitro were purified from human Gaucher type 1 and control spleen. Activator A1 (molecular mass 6,500 Da) had affinity for ConA-Sepharose, while activator A2 (molecular mass 3,500 Da) did not. Monospecific antibodies to each activator protein were prepared in rabbits by immunization with protein purified from type 1 Gaucher spleen. A1 and A2 activators from Gaucher type 1 spleen were shown to be immunochemically identical to A1 and A2 activators from control spleen. However, A1 and A2 activators, whether isolated from Gaucher type 1 or control spleen, were shown to be distinct proteins. Immunochemical examination of all collected fractions during the purification revealed the existence of a third activator (molecular mass 6,000 Da), which was antigenically identical to A1 activator but had no affinity for ConA-Sepharose. The two forms of A1 activator showed similar mobility on immunoelectrophoresis differing from that of A2 activator. Fibroblast extracts from controls and patients with different variants of Gaucher disease were investigated using immunodiffusion against antisera to A1 or A2 activator. In contrast to normal and Gaucher (types 1, 2 and 3) cell extracts, those of a Gaucher patient with normal glucosylceramidase activity had no visible precipitin line towards the antiserum against the two forms of A1 activator. The lack of crossreacting material to antibodies against A1 activator was confirmed by radial immunodiffusion and rocket immunoelectrophoresis. A1 activator stimulated the basal glucosylceramidase activity 5-6 fold in fibroblasts from this patient, whereas the normal effect was only a 1.2-1.5-fold stimulation. The immunological results together with the biochemical data provide evidence for the lack of an activator protein in a variant form of human Gaucher disease for the first time.     

Microsomal Triglyceride Transfer Protein Transfers and Determines Plasma Concentrations of Ceramide and Sphingomyelin but Not Glycosylceramide

Sphingolipids, a large family of bioactive lipids, are implicated in stress responses, differentiation, proliferation, apoptosis, and other physiological processes. Aberrant plasma levels of sphingolipids contribute to metabolic disease, atherosclerosis, and insulin resistance. They are fairly evenly distributed in high density and apoB-containing lipoproteins (B-lps). Mechanisms involved in the transport of sphingolipids to the plasma are unknown. Here, we investigated the role of microsomal triglyceride transfer protein (MTP), required for B-lp assembly and secretion, in sphingolipid transport to the plasma. Abetalipoproteinemia patients with deleterious mutations in MTP and absence of B-lps had significantly lower plasma ceramide and sphingomyelin but normal hexosylceramide, lactosylceramide, and different sphingosines compared with unaffected controls. Furthermore, similar differential effects on plasma sphingolipids were seen in liver- and intestine-specific MTP knock-out (L,I-Mttp^−/−) mice, suggesting that MTP specifically plays a role in the regulation of plasma ceramide and sphingomyelin. We hypothesized that MTP deficiency may affect either their synthesis or secretion. MTP deficiency had no effect on ceramide and sphingomyelin synthesis but reduced secretion from primary hepatocytes and hepatoma cells. Therefore, MTP is involved in ceramide and sphingomyelin secretion but not in their synthesis. We also found that MTP transferred these lipids between vesicles in vitro. Therefore, we propose that MTP might regulate plasma ceramide and sphingomyelin levels by transferring these lipids to B-lps in the liver and intestine and facilitating their secretion.