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.     

Metabolism of 1-pyrenedecanoic acid and accumulation of neutral fluorescent lipids in cultured fibroblasts of multisystemic lipid storage myopathy

The lipid metabolism in cultured fibroblasts from multisystemic (type 3) lipid storage myopathy and controls has been studied through pulse-chase experiments using 1-pyrenedecanoic acid as precursor. The uptake of 1-pyrenedecanoic acid was not significantly different in multisystemic lipid storage myopathy and control fibroblasts. The amount of fluorescent lipids synthesized by the cells was proportionally increasing with rising 1-pyrenedecanoic acid concentration in the culture medium. The proportion of the various fluorescent lipids does not significantly vary between 17 to 67 nmol/ml. But a 1-pyrenedecanoic acid concentration higher than 70-100 nmol/ml seems to be severely toxic for the cells. When incubated for 24 h in the presence of 1-pyrenedecanoic acid, at any concentration, the neutral lipid content (triacylglycerols, diacylglycerols and cholesterol esters) of cultured multisystemic lipid storage myopathy fibroblasts was higher than that of controls (around 600% of controls). Chase experiments showed that the biosynthesized triacylglycerols were not degraded in multisystemic lipid storage myopathy cells, but on the contrary were increased, probably by acylation of fluorescent fatty acids liberated from phospholipid turnover. In normal fibroblasts all the cellular fluorescence disappeared after 5 days chase and 1-pyrenedecanoic acid was recovered (as free 1-pyrenedecanoic acid) in the culture medium. In contrast, in multisystemic lipid storage myopathy fibroblasts, 40% of the fluorescence was remaining in the cells after 5 days chase; it was contributed by fluorescent triacylglycerols, which appeared as strongly fluorescent cytoplasmic vesicles. This probably results from a defect of the cytoplasmic catabolism of triacylglycerols which are accumulated in a cytoplasmic compartment independent of the lysosomal compartment (since the acid lysosomal lipase is not deficient in the multisystemic lipid storage myopathy cells). Finally, these results suggest a practical diagnostic application of 1-pyrenedecanoic acid, which can be used to differentiate multisystemic lipid storage myopathy from normal cultured fibroblasts.     

The effect of 3-methylindole on the rates of phospholipid and neutral lipid synthesis in cultured fibroblasts

The present study was designed to test the hypothesis that a pneumotoxin, 3-methylindole, alters the basic metabolic pathways involved in phospholipid and neutral lipid synthesis in cultured fibroblasts. Rat skin fibroblasts were obtained from day-old pups. Confluent monolayers were preincubated for up to 24 h with a range of concentrations (0-0.76 mM) of 3-methylindole. Following these treatments, the cell lipids were labelled by incubation for 6 h with [14C]glycerol. The lipids were extracted, separated by thin layer chromatography, and the radioactivity in each fraction was determined. 3-Methylindole had no effect on the total incorporation of [14C]glycerol into lipids, but significantly altered the distribution among lipid fractions. Incubation with 3-methylindole caused a decrease in the incorporation of [14C]glycerol into phosphatidylcholine, while radioactivity accumulated in the neutral lipid fraction. The other lipid fractions responded variably. Similarily, Flow 2000 human diploid lung fibroblasts were incubated for 24 h with 3-methylindole followed by treatment with [14C]glycerol, resulting in a 74% decrease in the incorporation of [14C]glycerol into phosphatidylcholine and a 50% increase in its accumulation in neutral lipid. The results indicate that 3-methylindole inhibits the synthesis of phosphatidylcholine from diacylglycerol precursors on the endoplasmic reticulum in cultured fibroblasts. This is an important observation as it shows that 3-methylindole affects the synthesis of phospholipids required for membrane turnover in cells that are not specialized for the production of phospholipids for surfactant.     

Reduction in apolipoprotein-mediated removal of cellular lipids by immortalization of human fibroblasts and its reversion by cAMP: lack of effect with Tangier disease cells.

High density lipoprotein (HDL) phospholipids and apolipoproteins remove cellular lipids by two distinct mechanisms, but their relative contribution to reverse cholesterol transport is unknown. Whereas phospholipid-mediated cholesterol efflux from cultured cells reflects the activity of the HDL receptor SR-BI, apolipoprotein-mediated lipid removal is regulated in response to changes in cellular cholesterol content (positive) and cell proliferation rates (negative). Here we show that immortalization of human skin fibroblast lines with the papillomavirus E6/E7 oncogenes increased their proliferation rates and selectively reduced the activity of the apolipoprotein-mediated lipid removal pathway. This reduction was accompanied by a decrease in cellular cAMP levels and was reversed by treatment with a cAMP analog. The stimulatory effect of cAMP was independent of changes in cellular phenotype or activities of cholesteryl ester cycle enzymes. The severely impaired apolipoprotein-mediated lipid removal pathway in Tangier disease fibroblasts, which persisted after immortalization, was not improved by treatment with a cAMP analog, implying that the cellular defect in Tangier disease is upstream from this cAMP-dependent signaling pathway.These results indicate that papillomavirus-induced immortalization of fibroblasts selectively reduces the activity of the apolipoprotein-mediated lipid removal pathway by a cAMP-dependent process, perhaps to prevent loss of cellular lipids needed for continual membrane synthesis.     

Lipid interactions with in vitro development of mammalian zygotes

Rabbit zygotes were tested for their ability to sequester radiolabeled acetate, oleate, and arachidonate in intracellular lipid. Radiolabeled arachidonic acid was concentrated 170 ± 28-fold (mean ± SEM) and oleic acid was concentrated 105 ± 26-fold in zygotic lipids during 6 hr of culture when compared with the initial concentrations in culture medium. Acetate was not concentrated into lipids by cultured zygotes. Both long chain fatty acids were incorporated mainly as triglyceride. Polydimethylsiloxane fluid, used to cover the microdroplets of medium during culture, demonstrated lipophilic properties. This characteristic was utilized to indirectly transfer lipids to culture medium, permitting examination only of lipoidal properties of test extracts on embryonal development. For rabbit zygotes, blood plasma extract was detrimental and whole blood extract was beneficial for embryonal cleavage rates during the first 24 hr of culture. A higher proportion of mouse zygotes developed to blastocysts when cultured in modified Ham's F-10 medium compared to BMOC medium, and this difference was negated by inclusion of a lipid extract prepared from rabbit oviductal fluid in the culture system. Comparison of fatty acid analyses of the lipid extracts with development rates of zygotes suggests that modified rates of embryo development may be associated with ratios of individual fatty acids presented to the culture medium rather than with the presence of any single fatty acid.     

The application of laser microprobe mass analysis to the study of biological material

Laser microprobe mass analysis (LAMMA) is an investigational method which is a powerful tool for the identification and quantitation of various elements present in small volumes of tissue. LAMMA is highly sensitive and capable of rapidly detecting concentrations of 1–3 p.p.m. of most metallic elements, in precisely localized cellular compartments. In order to further assess its value, cultured skin fibroblasts and biopsy tissues from human subjects and experimental animals were probed by LAMMA, and the results were correlated with ultrastructural findings. Biopsy samples were obtained from patients suffering from Gaucher disease, and from patients and animals with pathologic iron or copper metabolism. No significant abnormalities were detected in the cultured fibroblasts from patients with Gaucher disease, in contrast to the iron content of tissue biopsy Gaucher cells, which was markedly increased, apparently as a consequence of erythrophagocytosis. Particularly intense iron-related peaks were found in liver cytosiderosis due to neonatal or genetic haemochromatosis, thalassaemia major and in animal models of iron overload. An additional finding was the presence of aluminium accumulation in siderosomes of different cells. In liver biopsy samples from human Wilson's disease and from rats with an inherited disorder causing copper toxicosis, copper-containing compounds were identified and localized, and their relative concentration was estimated by LAMMA. The present study showed that LAMMA is a valuable technique for the localization and estimation of relative abundance of trace elements in various tissues containing excessive amounts of metals.     

Clinical variation in X-linked adrenoleukodystrophy: fatty acid and lipid metabolism in cultured fibroblasts

To determine whether the clinical phenotype of ALD correlates with the extent of metabolic abnormality, we investigated VLFA metabolism in cultured fibroblasts from patients with the clinically severe childhood from of ALD and the milder AMN variant. No differences were seen in the content of neutral lipids or phospholipids, in incorporation of [1-14C]lignocerate into cellular lipids, or in the fatty acid composition of fibroblasts from patients with childhood ALD or AMN. [1-14C]Lignocerate oxidation was deficient to a similar extent (35-40% of normal) in both intact fibroblasts and cell homogenates from patients with childhood ALD and AMN. With the use of fibroblast homogenates, oxidation of lignocerate was partially inhibited by various long-chain fatty acids, and residual activity in ALD homogenates was more susceptible to inhibition by palmitate than normal. In the presence of competing palmitate, residual lignocerate oxidative activity in fibroblast homogenates was reduced to 20 +/- 4% of normal in childhood ALD and 24 +/- 2% of normal in AMN. These results indicate that residual VLFA oxidative activity, fatty acid composition, VLFA metabolism, and lipid content of cultured fibroblasts do not correlate with the clinical expression of the ALD gene.     

Similar content of phospholipids and gangliosides in normal and homozygous familial hypercholesterolemia fibroblasts.

The cellular content of total and individual phospholipids and gangliosides was measured in fibroblasts cultured from four normal subjects, three patients with lysosomal lipid storage diseases, and two subjects with homozygous familial hypercholesterolemia. Measurements were made on cells grown in medium containing fetal calf serum under conditions in which normal cells derive cholesterol for cell growth from low density lipoprotein present in the fetal calf serum, whereas familial hypercholesterolemia homozygote cells, which lack cell surface low density lipoprotein receptors, derive cholesterol from endogenous synthesis. No difference was observed in the cellular content of total or individual phospholipids and gangliosides in the normal and familial hypercholesterolemia homozygote cells. In contrast, cells from a patient with Niemann-Pick disease and a patient with Sandhoff disease showed elevations in the content of sphingomyelin and complex gangliosides, respectively.     

Increased affinity of liposomes derived from total spleen and liver lipids to spleen cells

The interaction of liposomes derived from total lipids of mouse spleen and liver with mouse spleen cells was studied. It was shown that the binding of these liposomes is much higher than the binding of liposomes obtained from a model lipid mixture--phosphatidylcholine--phosphatidylethanolamine--cholesterol (2:1:1). Adherent and nonadherent spleen cells were found to have affinity for liposomes derived from total lipids of spleen or liver. Removal of gangliosides and protein contaminants from the liposomes derived from total spleen lipids caused an increased binding of liposomes to spleen cells. Multilamellar liposomes bound more effectively to ultrasonicated vesicles having a homologous lipid composition than the liposomes with a different lipid composition. The increased affinity of liposomes derived from total lipids of spleen or liver for spleen cells may account for the identical fluidity of the lipid bilayer of liposomes and plasma membranes of spleen cells.     

Analysis of lipid-composition changes in plasma membrane microdomains

Sphingolipids accumulate in plasma membrane microdomain sites, such as caveolae or lipid rafts. Such microdomains are considered to be important nexuses for signal transduction, although changes in the microdomain lipid components brought about by signaling are poorly understood. Here, we applied a cationic colloidal silica bead method to analyze plasma membrane lipids from monolayer cells cultured in a 10 cm dish. The detergent-resistant fraction from the silica bead-coated membrane was analyzed by LC-MS/MS to evaluate the microdomain lipids. This method revealed that glycosphingolipids composed the microdomains as a substitute for sphingomyelin (SM) in mouse embryonic fibroblasts (tMEFs) from an SM synthase 1/2 double KO (DKO) mouse. The rate of formation of the detergent-resistant region was unchanged compared with that of WT-tMEFs. C2-ceramide (Cer) stimulation caused greater elevations in diacylglycerol and phosphatidic acid levels than in Cer levels within the microdomains of WT-tMEFs. We also found that lipid changes in the microdomains of SM-deficient DKO-tMEFs caused by serum stimulation occurred in the same manner as that of WT-tMEFs. This practical method for analyzing membrane lipids will facilitate future comprehensive analyses of membrane microdomain-associated responses.     

The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes

This study aimed to investigate the use of Nile red, a fluorescent dye specific for intracellular lipid droplets, to quantify the lipid content of single mammalian oocytes. It was hypothesized that a higher amount of lipid present in lipid droplets in an oocyte would result in a higher amount of emitted fluorescent light. Following fixation and subsequent staining of denuded oocytes, the fluorescence of the whole oocyte was visualized by fluorescence microscopy and quantified with a photometer and photomultiplier connected to the microscope. The peak of fluorescence was observed in the yellow spectrum (590 nm) and the fluorescence was restricted to the lipid droplets corresponding to apolar lipids. Nile red concentrations ranging from 0.1 to 10 microg/ml yielded similar results. After fixation, a minimum of 2 h staining was necessary to reach maximal fluorescence which remained stable for several hours. The position of the microscopic focus within the oocyte had no influence on the amount of measured fluorescence. Successive measurements of the same oocyte yielded very similar results indicating the repeatability of the method. Finally, the technique was validated by comparing the lipid content of bovine, porcine and murine immature oocytes, which are known to contain different amounts of lipids. After staining, the fluorescence of murine oocytes was 2.8-fold lower than the fluorescence of bovine oocytes which in turn were 2.4 times less fluorescent than porcine oocytes. Based on this study, it can be said that this rather fast and easy technique allows for the relative quantification of the lipid content (present in the lipid droplets) of one single oocyte. The different amounts of emitted fluorescent light in bovine, porcine and murine oocytes correlated with the known lipid contents in these three species. This technique could be used to compare the lipid content of oocytes originating from different donors, from different sized follicles or cultured in various conditions.     

Biochemical and ultrastructural features of human fibroblasts cultured from a new variant of type 3 lipid storage myopathy

A new variant of multisystemic lipid storage myopathy (type 3) has been identified. Human cultured fibroblasts present a major triacylglycerol storage whereas other neutral lipids and phospholipids are in the normal range. When feeding the cells in the presence of radiolabelled oleic acid we observed an accumulation of radiolabelled triacylglycerols demonstrating the endogenous biosynthesis of the stored triacylglycerols. After a 72-hr chase period, no degradation of radiolabelled triacylglycerols was observed. Histochemical examination of multisystemic lipid storage myopathy skin fibroblasts showed a massive accumulation of neutral lipids (stained by the fluorescent probe Nile Red) in cells grown in medium supplemented with 10% fetal calf serum. These cytoplasmic vacuoles were not obviously membrane-surrounded as shown by electron microscopy.     

In utero diagnosis of Gaucher disease.

Beta-Glucosidase activity measured by synthetic substrate at pH 4.6 was low in the cultured amniotic cells from two pregnant women at risk for juvenile and adult type Gaucher disease. The diagnosis was confirmed by showing a low activity of beta-glucosidase in the skin fibroblasts with a synthetic substrate or in the spleen with a natural substrate, and by ascertaining the presence of Gaucher cells in the fetal tissues. However, considerable activity of beta-glucosidase measured with synthetic substrate was found in the liver of both affected fetuses and in the spleen of one. It is advisable that the determination of beta-glucosidase to confirm prenatal diagnosis of Gaucher disease be done either in the cultured skin fibroblasts or in the spleen, and if in the spleen, with a natural substrate rather than a synthetic one.     

Occurrence of J blood group activities in lipid and non-lipid fractions of organs and body fluids of cattle

Total lipids and protein-containing residues obtained after lipid extraction from various organs and body fluids of J-positive cattle were tested for J activity in the bovine J blood group system. Polar lipids prepared by column chromatography of total lipids, which contain predominantly neutral lipids, were also tested. Total lipids (or polar lipids, respectively) were analysed for lipid phosphorus, lipid sugar, and hexosamine. Both lipids and non-lipid fractions of brain, myocardium, skeletal muscle, and adipose tissue show no J activity. The lipids of urinary bladder epithelium, spleen, liver, and kidney are J-positive, whereas their non-lipid fractions are J-negative. Both the lipids and the non-lipid fractions of seminal plasma, spermatozoa, and faeces are J-active. The lipids extracted from hair show no J activity, while those of cornea and eyelens are J-active. The high amount of glycolipids from seminal plasma, spermatozoa and spleen stimulates further studies of these lipids.     

Comparison of the intracellular lipids of cultured vascular endothelial cells and fibroblasts

Summary The lipids of human umbilical vein endothelial cells, calf aortic endothelial cells and foreskin fibroblasts have been compared. Cell cultures were established, and, upon confluency, the lipids were extracted and analyzed with respect to total lipid content, classes of lipids and total lipid fatty acid composition. The total quantity of lipid per milligram protein found in both human umbilical vein endothelium and calf aorta endothelium was similar to that found in fibroblasts grown in similar medium. Both types of endothelium contained the same major neutral lipid classes as fibroblasts, although they contained more phospholipid than did fibroblasts. The fatty acid composition of the three cells examined was influenced by cell type as well as the type of serum in the culture medium. This work was supported by PHS Grants HL16058, HL19638, AM14626 and HL18827.     

Top-down lipidomics of low density lipoprotein reveal altered lipid profiles in advanced chronic kidney disease

This study compared the molecular lipidomic profile of LDL in patients with nondiabetic advanced renal disease and no evidence of CVD to that of age-matched controls, with the hypothesis that it would reveal proatherogenic lipid alterations. LDL was isolated from 10 normocholesterolemic patients with stage 4/5 renal disease and 10 controls, and lipids were analyzed by accurate mass LC/MS. Top-down lipidomics analysis and manual examination of the data identified 352 lipid species, and automated comparative analysis demonstrated alterations in lipid profile in disease. The total lipid and cholesterol content was unchanged, but levels of triacylglycerides and N-acyltaurines were significantly increased, while phosphatidylcholines, plasmenyl ethanolamines, sulfatides, ceramides, and cholesterol sulfate were significantly decreased in chronic kidney disease (CKD) patients. Chemometric analysis of individual lipid species showed very good discrimination of control and disease sample despite the small cohorts and identified individual unsaturated phospholipids and triglycerides mainly responsible for the discrimination. These findings illustrate the point that although the clinical biochemistry parameters may not appear abnormal, there may be important underlying lipidomic changes that contribute to disease pathology. The lipidomic profile of CKD LDL offers potential for new biomarkers and novel insights into lipid metabolism and cardiovascular risk in this disease.     

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease¹. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms² and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson''s disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.     

25-OHD3-26,23 lactone: demonstration of kidney-dependent synthesis in the pig and rat

At late confluency (21 days after passage), cultured skin fibroblasts from G_M1 gangliosidosis, type 1 patients showed approximately a 15-fold increase in G_M1 ganglioside, and fibroblasts from Tay-Sachs and Sandhoff disease patients showed a 50- and 30-fold increase in G_M2 ganglioside, respectively, when compared to normal fibroblasts. Since demonstration of storage material is important for accurate diagnosis of the lysosomal storage disorders, analysis of the accumulating lipids in late confluency fibroblasts can provide an additional tool for the diagnosis of the gangliosidoses and possibly other lysosomal disorders.     

Free sphingoid bases in tissues from patients with type C Niemann-Pick disease and other lysosomal storage disorders

The 20-fold increase of free sphingoid bases found in liver from a murine model of Niemann-Pick type C (NPC) combined to the NPC-like phenotype induced by addition of sphinganine to normal fibroblast cultures prompted us to investigate the potential involvement of these compounds in the human disease. The contents of sphingosine and sphinganine were measured in liver, spleen, brain and skin fibroblast cultures by a sensitive HPLC method. In liver and spleen from NPC patients, a 6- to 24-fold elevation of sphingosine and sphinganine already prominent at the fetal stage of the disease was observed, while no clear increase could be evidenced in brain tissue. A significant increase, not modulated by the intralysosomal content of free cholesterol, also occurred in skin fibroblast cultures. To investigate the specificity of these findings, other lysosomal storage disorders were studied. A striking accumulation was found in liver and spleen (24- to 36-fold) from patients with Niemann-Pick disease type A and B (sphingomyelinase-deficient forms), and in cerebral cortex of type A Niemann-Pick disease. A significant storage also occurred in Sandhoff disease, while several other sphingolipidoses showed a moderate elevation. In all cases but Sandhoff disease brain, the sphingosine/sphinganine ratio remained unchanged, suggesting that the accumulated free sphingoid bases derived from sphingolipid catabolism. Formation of complexes between sphingosine and the lipid material accumulated in lysosomes might be a general mechanism in lysosomal lipidoses. In NPC, however, an increase of free sphingoid bases disproportionate to the degree of lysosomal storage and a specific involvement of cultured fibroblasts suggested a more complex or combined mechanism.     

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.