Decreased sphingomyelin (t34:1) is a candidate predictor for lung squamous cell carcinoma recurrence after radical surgery: a case-control study.

BACKGROUND: To reduce disease recurrence after radical surgery for lung squamous cell carcinomas (SQCCs), accurate prediction of recurrent high-risk patients is required for efficient patient selection for adjuvant chemotherapy. Because treatment modalities for recurrent lung SQCCs are scarce compared to lung adenocarcinomas (ADCs), accurately selecting lung SQCC patients for adjuvant chemotherapy after radical surgery is highly important. Predicting lung cancer recurrence with high objectivity is difficult with conventional histopathological prognostic factors; therefore, identification of a novel predictor is expected to be highly beneficial. Lipid metabolism alterations in cancers are known to contribute to cancer progression. Previously, we found that increased sphingomyelin (SM)(d35:1) in lung ADCs is a candidate for an objective recurrence predictor. However, no lipid predictors for lung SQCC recurrence have been identified to date. This study aims to identify candidate lipid predictors for lung SQCC recurrence after radical surgery. METHODS: Recurrent (n = 5) and non-recurrent (n = 6) cases of lung SQCC patients who underwent radical surgery were assigned to recurrent and non-recurrent groups, respectively. Extracted lipids from frozen tissue samples of primary lung SQCC were analyzed by liquid chromatography-tandem mass spectrometry. Candidate lipid predictors were screened by comparing the relative expression levels between the recurrent and non-recurrent groups. To compare lipidomic characteristics associated with recurrent SQCCs and ADCs, a meta-analysis combining SQCC (n = 11) and ADC (n = 20) cohorts was conducted. RESULTS: Among 1745 screened lipid species, five species were decreased (≤ 0.5 fold change; P < 0.05) and one was increased (≥ 2 fold change; P < 0.05) in the recurrent group. Among the six candidates, the top three final candidates (selected by AUC assessment) were all decreased SM(t34:1) species, showing strong performance in recurrence prediction that is equivalent to that of histopathological prognostic factors. Meta-analysis indicated that decreases in a limited number of SM species were observed in the SQCC cohort as a lipidomic characteristic associated with recurrence, in contrast, significant increases in a broad range of lipids (including SM species) were observed in the ADC cohort. CONCLUSION: We identified decreased SM(t34:1) as a novel candidate predictor for lung SQCC recurrence. Lung SQCCs and ADCs have opposite lipidomic characteristics concerning for recurrence risk. TRIAL REGISTRATION: This retrospective study was registered at the UMIN Clinical Trial Registry ( UMIN000039202 ) on January 21, 2020.

Obese Mice with Dyslipidemia Exhibit Meibomian Gland Hypertrophy and Alterations in Meibum Composition and Aqueous Tear Production.

BACKGROUND: Dyslipidemia may be linked to meibomian gland dysfunction (MGD) and altered meibum lipid composition. The purpose was to determine if plasma and meibum cholesteryl esters (CE), triglycerides (TG), ceramides (Cer) and sphingomyelins (SM) change in a mouse model of diet-induced obesity where mice develop dyslipidemia. METHODS: Male C57/BL6 mice (8/group, age = 6 wks) were fed a normal (ND; 15% kcal fat) or an obesogenic high-fat diet (HFD; 42% kcal fat) for 10 wks. Tear production was measured and meibography was performed. Body and epididymal adipose tissue (eAT) weights were determined. Nano-ESI-MS/MS and LC-ESI-MS/MS were used to detect CE, TG, Cer and SM species. Data were analyzed by principal component analysis, Pearson's correlation and unpaired t-tests adjusted for multiple comparisons; significance set at p ≤ 0.05. RESULTS: Compared to ND mice, HFD mice gained more weight and showed heavier eAT and dyslipidemia with higher levels of plasma CE, TG, Cer and SM. HFD mice had hypertrophic meibomian glands, increased levels of lipid species acylated by saturated fatty acids in plasma and meibum and excessive tear production. CONCLUSIONS: The majority of meibum lipid species with saturated fatty acids increased with HFD feeding with evidence of meibomian gland hypertrophy and excessive tearing. The dyslipidemia is associated with altered meibum composition, a key feature of MGD.

Phase diagram of a polyunsaturated lipid mixture: Brain sphingomyelin/1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine/cholesterol.

Phospholipids having a polyunsaturated acyl chain are abundant in biological membranes, but their behavior in lipid mixtures is difficult to study. Here we elucidate the nature of such mixtures with this report of the first ternary phase diagram containing the polyunsaturated lipid SDPC in mixtures of BSM/SDPC/Chol (brain sphingomyelin/1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine/cholesterol). These mixtures show coexisting macroscopic liquid-disordered (Ld) and liquid-ordered (Lo) phase separation, with phase boundaries determined by FRET and by fluorescence microscopy imaging of giant unilamellar vesicles (GUVs). Surprisingly, SDPC mixes with BSM/Chol similarly to how DOPC and POPC mix with BSM/Chol. Notably, intermediate states are produced within the Ld+Lo liquid-liquid immiscibility region upon addition of fourth component POPC. These mixtures of BSM/SDPC/POPC/Chol exhibit nanoscopic Ld+Lo domains over a very large volume of composition space, possibly because Ld/Lo line tension is not high.

Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry.

Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1µm, and larger vesicles of around 10-µm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.

Unique thermal behavior of sphingomyelin species with nonhydroxy and 2-hydroxy very-long-chain (C28-C32) PUFAs.

In rat germ cells and spermatozoa, sphingomyelin (SM) contains molecular species with nonhydroxy (n) and 2-hydroxy (h) very-long-chain polyunsaturated fatty acids (V), the most abundant being SMs with (n- and h-) 28:4n-6, 30:5n-6, and 32:5n-6 as acyl chains. The aim of this study was to gain information about their thermotropic behavior and interactions with other lipids. After isolation from rat testis, multilamellar and giant unilamellar vesicles from these SMs were examined using fluorescent probes. Only n-32:5 SM and h-32:5 SM displayed a gel-liquid transition temperature (Tt ∼ 21-22°C), the rest remaining in the liquid state in the 5°C-45°C range. The degree of order was larger in bilayers of any of the h-V SMs than in those of their chain-matched n-V SMs. Both, but n-V SM relatively more than h-V SM, decreased the Tt of dimyristoylphosphatidylcholine as their proportion increased in binary phosphatidylcholine:SM liposomes. In contrast to the established ability of 16:0 SM to form lateral cholesterol/SM-rich ordered domains in ternary dioleoylphosphatidylcholine:cholesterol:SM bilayers, neither n-V SM nor h-V SM showed a tendency to do so. Thus, these SMs are in the fluid state and are not involved in this type of domains in spermatozoa at physiological temperatures. However, this state could be altered at the very low temperatures at which these gametes are usually preserved.

Unique sphingomyelin patches are targets of a beta-cell-specific antibody.

To devise successful imaging and therapeutic strategies, the identification of ß-cell surface markers is one of the challenges in diabetes research that has to be resolved. We previously showed that IC2, a rat monoclonal IgM antibody, can be used for ex vivo determination of ß-cell mass by imaging. Further progress toward the development of an antibody-based imaging agent was hampered by the lack of knowledge regarding the nature and composition of the IC2 antigen. Here, we show a series of systematic experiments involving classical lipid extraction and chromatography techniques combined with immunochemistry, which led to the identification of sphingomyelin as the target antigen assembled in the form of patches on the ß-cell surface. Our findings were verified by modulating SM by enzymatic cleavage, downregulation, upregulation, and perturbation of membrane SM and observation of corresponding changes in IC2 binding. Cholesterol participates in stabilization of these patches, as its removal results in loss of IC2 binding. We believe that these findings have implications for identifying future ligands for the proposed antigen for imaging purposes as well as for potential therapy, as sphingomyelin has been shown to play a role in the apoptotic cascade in pancreatic ß cells.

Simultaneous quantification of the lipids phosphatidylcholine, 3-sn-phosphatidylethanolamine, sphingomyelin, and L-α-lysophosphatidylcholine extracted from the tissues of the invasive golden apple snail (Pomacea canaliculata) using UHPLC-ESI-MS/MS.

Lipids such as phosphatidylcholine (PC), 3-sn-phosphatidylethanolamine (PE), sphingomyelin (SM) and L-α-lysophosphatidylcholine (LPC) are the major components of biological membranes and play important roles in physiological functions. Here, PC, PE, SM, and LPC were extracted from golden apple snails (GAS, Pomacea canaliculata) and GAS flesh (GASF) using an ethanol/hexane sequential scheme and quantified simultaneously using ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) to evaluate whether the GAS could be the source of the four lipids. Our results suggest that ethanol extracts contained the most crude lipids, and the yield of dry (evaporated) lipids were 3.45 g per 100 g fresh GASF and 1.82 g per 100 g of fresh GAS. Quantification of the lipids using UHPLC-ESI-MS/MS suggested that GAS contained PE, PC, SM and LPC, with SM being the most abundant lipid (after purification: 1.71 and 1.42 mg g-1 dry weight from 100 g of GASF and GAS, respectively). The method we used is cost-effective, and the recovery rates of ethanol and hexane ranged from 80-91% and 87-91% respectively. Overall, GAS and GASF are potential raw materials for lipids such as SM and PC extraction using the ethanol/hexane method. Comparatively, lipids extraction from the GAS is more effective and timesaving. Our finding would provide a way to utilize GAS and potentially control its invasion.

UHPLC-Q-Orbitrap HRMS-based quantitative lipidomics reveals the chemical changes of phospholipids during thermal processing methods of Tan sheep meat.

Thermal processing affects the lipid compositions of meat products. The study determined the effects of boiled, steamed and roasted processing methods on the lipidomics profiles of Tan sheep meat with a validated UPLC-Q-Orbitrap HRMS combined lipid screening strategy method. Combined with sphingolipid metabolism, the boiled approach was the suitable choice for atherosclerosis patients for more losses of sphingomyelin than ceramide in meat. The similarly less losses of phosphatidylcholine and lysophosphatidylcholine showed in glycerophospholipid metabolism implied that steamed Tan sheep meat was more suitable for the populations of elderly and infants. Furthermore, a total of 90 lipids with significant difference (VIP > 1) in 6 lipid subclasses (sphingomyelin, ceramide, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamines, triacylglycerol,) were quantified among raw and three types of thermal processed Tan sheep meat, further providing useful information for identification of meat products with different thermal processing methods (LOD with 0.14-0.31 µg kg-1, LOQ with 0.39-0.90 µg kg-1).

Developmentally regulated sphingolipid synthesis in African trypanosomes.

Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream-stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labelling confirmed stage-specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased more than threefold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six-transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian sphingomyelin synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.

Isolation and characterization of luminal membranes from urinary bladder.

A method is reported for the isolation of a highly purified fraction of urinary bladder membranes containing hexagonal plaques. The method uses zonal centrifugation as the final step of fractionation. The purified fraction was characterized by its electron microscopic morphology, by its enzymatic profile, by quantitative and qualitative analysis of lipids and by the protein pattern obtained by electrophoresis in polyacrylamide sodium dodecyl sulfate gels. The fraction contains 65% lipids and 35% proteins. The major protein component has a molecular weight of 27,000 daltons. Phospholipids are more than the 54% of the total lipid weight. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol are the major phospholipids with 50%, 30%, and 7% of the total lipid phosphorus, respectively. The glycolipid fraction is 10% of the total lipid weight and is formed by only two components, both sulfatides. Total cholesterol makes up 36% of the total neutral lipid fraction of which cholesterol esters constitute 6%. Glycoproteins are also found to be present in the fraction.

Simultaneous preparation of purified plasmalogens and sphingomyelin in human erythrocytes with phospholipase A1 from Aspergillus orizae.

A method for the simultaneous purification of plasmalogens and sphingomyelin (SM) in human erythrocytes is described. Treatment of total lipids with n-hexane/acetone (1:1 v/v) resulted in selective precipitation of SM. Both the supernatant and the precipitate fractions were incubated with a phospholipase A(1) (PLA1) from Aspergillus orizae for 3.5 h. The PLA1-treated lipids were extracted with n-hexane/isopropanol, the hexane layer was obtained using a Na(2)SO(4) solution, and the hexane layer was further washed with water. At this step, the relative concentration of the plasmalogens was 92% of the total phospholipids in the supernatant fraction, and that of SM was 97.7% in the precipitate fraction. Each fraction was applied to high performance liquid chromatography (HPLC) for further purification. The plasmalogen and SM obtained were almost free of the other lipids. The purity of the plasmalogens and SM was monitored by HPLC, which can separate intact plasmalogens from their diacyl analogs.

A Targeted Mass Spectrometric Analysis Reveals the Presence of a Reduced but Dynamic Sphingolipid Metabolic Pathway in an Ancient Protozoan, Giardia lamblia.

Giardia lamblia, a single-celled eukaryote, colonizes and thrives in the small intestine of humans. Because of its compact and reduced genome, Giardia has adapted a "minimalistic" life style, as it becomes dependent on available resources of the small intestine. Because Giardia expresses fewer sphingolipid (SL) genes-and glycosphingolipids are critical for encystation-we investigated the SL metabolic cycle in this parasite. A tandem mass spectrometry (MS/MS) analysis reveals that major SLs in Giardia include sphingomyelins, sphingoid bases, ceramides, and glycosylceramides. Many of these lipids are obtained by Giardia from the growth medium, remodeled at their fatty acyl chains and end up in the spent medium. For instance, ceramide-1-phosphate, a proinflammatory molecule that is not present in the culture medium, is generated from sphingosine (abundant in the culture medium) possibly by remodeling reactions. It is then subsequently released into the spent medium. Thus, the secretion of ceramide-1-phospate and other SL derivatives by Giardia could be associated with inflammatory bowel disease observed in acute giardiasis. Additionally, we found that the levels of SLs increase in encysting Giardia and are differentially regulated throughout the encystation cycle. We propose that SL metabolism is important for this parasite and, could serve as potential targets for developing novel anti-giardial agents.

Deep Profiling of Immunosuppressive Glycosphingolipids and Sphingomyelins in Wild Cordyceps.

Deep profiling of glycosphingolipids and sphingomyelins in wild Cordyceps was carried out by using offline chromatographic enrichment followed by ultrahigh performance liquid chromatography-ultrahigh definition-quadrupole time-of-flight mass spectrometry (UHPLC-UHD-Q-TOF-MS). A total of 119 glycosphingolipids (72 new ones) and 87 sphingomyelins (43 new ones) were identified from wild Cordyceps on the basis of the accurate mass and MS/MS fragmentations, isotope patterns, sphingolipid (SPL) database matching, confirmation by SPL standards, and the reversed-phase liquid chromatographic retention rule. This study is the most comprehensive report on the identification of glycosphingolipids and sphingomyelins from fungus. A subsequent lipopolysaccharide-induced mouse splenic lymphocyte proliferation assay showed that the Cordyceps glycosphingolipid fraction exhibits higher immunosuppressive activity compared to that of Cordyceps sphingomyelins. Our findings provided insight into the chemical diversity of sphingolipids in Cordyceps and chemical evidence for the therapeutic application of wild Cordyceps.

The isolation and structure of membrane lipid rafts from rat brain.

The action of detergents in the isolation of detergent-resistant membrane fractions from rat brain is reported. Triton X-100 treatment of whole rat brain homogenate at 4 degrees C produced detergent-resistant membranes with a density of 1.07g/ml compared with Brij96 where the density of the membrane was only 1.05g/ml. The DRM fractions isolated using Triton X-100 are considerably heavier than those isolated from homogenates treated with Brij96. The major polar lipid composition of DRMs derived from Brij96 treated homogenates have a higher proportion of aminophospholipids compared with choline phospholipids than Triton X-100 derived DRMs; this may indicate that DRMs from Brij96 treated homogenates are more closely related to the parent membrane in lipid composition. Solubilization by Triton X-100 at higher temperatures resulted in the appearance of a second detergent-resistant membrane fraction distinctly lighter in density than the membrane recovered at density 1.07g/ml. Analysis of phospholipid composition of the brain homogenate during detergent treatment for up to 30min at 37 degrees C showed a decreasing proportion of sphingomyelin. Treatment of homogenates at 37 degrees C appears to activate phospholipases/sphingomyelinases that may alter the lipid content of isolated DRMs. The presence of K+/Mg2+ with Brij96 treatment results in DRM fractions with significantly thicker bilayers and of larger vesicle diameter than DRMs isolated from either Triton X-100 or Brij96 treated homogenates in the absence of cations.

Method to Measure Sphingomyelin Synthase Activity Changes in Response to CD95L.

Sphingomyelin synthases 1 and 2 convert the anti-oncometabolite ceramide to sphingomyelin, the most abundant sphingolipid in plasma membrane. CD95L-induced ceramide increase is associated with the caspase-dependent inhibition of sphingomyelin synthesis, which enhances the mitochondrial route to apoptosis. Knocking down sphingomyelin synthase 1 or inhibiting sphingomyelin synthesis facilitates ceramide accumulation, cytochrome c release from mitochondria, and caspase-9 activation in cancer cell upon CD95L treatment. Here, we describe a method to monitor in situ sphingomyelin synthase activity changes triggered by CD95L.

Endometriosis foci differentiation by rapid lipid profiling using tissue spray ionization and high resolution mass spectrometry.

Obtaining fast screening information on molecular composition of a tissue sample is of great importance for a disease biomarkers search and for online surgery control. In this study, high resolution mass spectrometry analysis of eutopic and ectopic endometrium tissues (90 samples) is done using direct tissue spray mass spectrometry in both positive and negative ion modes. The most abundant peaks in the both ion modes are those corresponding to lipids. Species of three lipid classes are observed, phosphatidylcholines (PC), sphingomyelins (SM) and phosphoethanolamines (PE). Direct tissue analysis gives mainly information on PC and SM lipids (29 species) in positive ion mode and PC, SM and PE lipids (50 species) in negative ion mode which gives complementary data for endometriosis foci differentiation. The biggest differences were found for phospholipids with polyunsaturated acyls and alkils. Although, tissue spray shows itself as appropriate tool for tissue investigation, caution should be paid to the interpretation of mass spectra because of their higher complexity with more possible adducts formation and multiple interferences must be taken into account. The present work extends the application of direct tissue analysis for the rapid differentiation between endometriotic tissues of different foci.

Effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine on membrane lipid profile and cryotolerance of human sperm.

OBJECTIVE: To study the effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine (soy-PC) on sperm cryotolerance with regard to sperm membrane lipid profile, membrane surface integrity, and routine semen parameters. DESIGN: Experimental study. SETTING: University-affiliated tertiary hospital. PATIENT(S): A total of 20 normospermic fertile men. INTERVENTION(S): Semen samples examined for differences in semen parameters, sperm membrane lipid profile, and plasma membrane surface both before and after cryopreservation using basic freezing medium with N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) and tris-(hydroxymethyl)-aminomethane (TRIS) supplemented with purified soy-PC (TEST-PC) or egg yolk (TEST-Y), both alone or in association (TEST-Y-PC). MAIN OUTCOME MEASURE(S): Conventional semen parameters and membrane lipid profile by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). RESULT(S): Postthaw sperm cell motility, vitality, and morphology parameters were similar for soy-PC (TEST-PC) and egg yolk (TEST-Y) cryoprotectants. However, sperm exposed to TEST-Y-PC presented better kinetic parameters, which were similar to the original quality of the fresh semen. Human sperm MALDI-MS lipid profiles revealed that the relative abundance of glycerophospholipids of m/z 760.44 [PC (34:1)+H]+, 781.55 [SM (20:0) +Na]+, 784.55 [PC (36:3) +H]+, 806.64 [PC (38:6) +H]+, 807.64 [SM (22:1) +Na]+, and 809.64 [SM (22:0) +Na]+ increased in soy-PC samples (TEST-PC). Nonetheless, only one lipid (m/z 781.55, [SM (20:0) +Na]+) statistically significantly changed when sperm was cryopreserved in TEST-Y-PC. CONCLUSION(S): Sphingomyelin was defined as a prospective biomarker of soy-PC treatment, and it could be related to the positive cryoprotective effects of soy-PC in human sperm, opening new perspectives to design of a more efficient synthetic cryoprotectant medium containing purified egg yolk biomolecules combined with soy-PC.

2H- and 31P-NMR studies of cholesteryl palmitate in sphingomyelin dispersions.

Dispersions (50 wt% in water) of sphingomyelin/cholesteryl palmitate (95 : 5 mol%) have been studied by 2H- and 31P-NMR spectroscopy between 25 and 60 degrees C. The deuterated esters, cholesteryl palmitate-d31 and cholesteryl palmitate-16, 16, 16-d3, were used for 2H-NMR studies. Of the 5 mol% cholesteryl palmitate added, 1.5 mol% was found to incorporate in the sphingomyelin and this amount remained constant throughout the temperature range studied. The remainder of the cholesteryl palmitate was present as regions of solid. At temperatures above the gel-to-liquid crystalline phase transition of sphingomyelin the NMR spectra indicate that a fraction of the cholesteryl palmitate and sphingomyelin is undergoing rapid isotropic motions. This fraction, which increases with increasing temperature above the phase transition, is probably composed of small bilayer structures. When 50 mol% cholesterol (relative to sphingomyelin) was added to the sphingomyelin/cholesteryl palmitate dispersion, the isotropic component in the NMR spectral was no longer present, the gel-to-liquid crystalline phase transition was removed, and the incorporation of ester in the membrane decreased by more than an order of magnitude.

Biochemical studies in Niemann-Pick disease. I. Major sphingolipids of liver and spleen.

In liver and spleen specimens of 12 patients with Niemann-Pick disease types A or B, sphingomyelin was increased 15-45-fold, total phospholipids 4-10-fold and cholesterol 3-6-fold over the normal values. The storage pattern was qualitatively similar in both types but the degree of accumulation was less in type B. In Niemann-Pick disease type C (16 cases), sphingomyelin was increased 3.5-fold in liver and 6-fold in spleen. In all forms of Niemann-Pick disease, bis(monoacylglycero)phosphate was markedly elevated. Glycosphingolipids were studied in six cases with type C, three cases with type B and two cases with type A. Glucosylceramide showed the largest increase from the normal pattern in all types of Niemann-Pick disease. Highest values were recorded in type C, 14- and 35-fold normal concentrations in liver and spleen, respectively. Other neutral glycosphingolipids, particularly lactosylceramide, were also elevated, and a 2-4-fold increase of ganglioside GM3 occurred. The fatty acid profiles of the sphingolipids showed only minor alterations. In contrast to the largely dominating sphingomyelin storage found in liver and spleen of Niemann-Pick disease types A and B, the major characteristic of the lipid storage in Niemann-Pick disease type C was the absence of any prevailing accumulation and, thus, the concept of this disorder as a primary sphingomyelin storage disease is not founded.

Clear differences in ceramide metabolism between glycosphingolipids and sphingomyelin in a human promyelocytic leukemia cell line HL-60 stimulated by a differentiation inducer.

Although the ceramide components of both glycosphingolipids (GSLs) and sphingomyelin (SM) in HL-60 cells were identical, the molecular species of the ceramides preferentially used in biosynthesis were quite different in GSLs and SM. When HL-60 cells were stimulated to differentiate into macrophage-like cells by phorbol ester after their sphingolipids had been metabolically labeled with L-[3-14C]serine to saturation point, marked changes in the radioactivities of the ceramide residues were observed in GSLs, showing the activation of a biosynthetic pathway of ganglioside GM3. No significant changes were, however, observed in the ceramide residues of SM. These results indicate that it is necessary to consider the overall metabolism of ceramides, including their origin, when investigating the functions of ceramides in signal transduction systems.