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.     

Basic fibroblast growth factor-induced proliferation of primary astrocytes. evidence for the involvement of sphingomyelin biosynthesis

We recently reported that the marked decrease in cellular ceramide in primary astrocytes is an early event associated with the mitogenic activity of basic fibroblast growth factor (bFGF) (Riboni, L., Viani, P., Bassi, R., Stabieini, A., and Tettamanti, G. (2000) GLIA 32, 137-145). Here we show that a rapid activation of sphingomyelin biosynthesis appears to be the major mechanism responsible for the fall in ceramide levels induced by bFGF. When quiescent astrocytes were treated with bFGF, an increased amount of newly synthesized ceramide (from either l-[(3)H]serine or [(3)H]sphingosine) was directed toward the biosynthesis of sphingomyelin. Conversely, bFGF did not appear to affect ceramide levels by other metabolic pathways involved in ceramide turnover such as sphingomyelin degradation and ceramide biosynthesis, degradation, and glucosylation. Enzymatic studies demonstrating a relevant and rapid increase in sphingomyelin synthase activity after bFGF treatment have provided a convincing explanation for the activation of sphingomyelin biosynthesis. The bFGF-induced increase in sphingomyelin synthase appears to depend on a post-translational activation mechanism. Moreover, in the presence of brefeldin A, the activation of sphingomyelin biosynthesis was abolished, suggesting that the enzyme is located in a compartment other than the Golgi apparatus. Also the phosphatidylcholine-specific phospholipase C inhibitor D609 exerted a potent inhibitory effect on sphingomyelin biosynthesis. Finally, we demonstrate that inhibition of sphingomyelin biosynthesis by brefeldin A or D609 led to a significant inhibition of bFGF-stimulated mitogenesis. All this supports that, in primary astrocytes, the early activation of sphingomyelin synthase is involved in the bFGF signaling pathway leading to proliferation.     

Sphingomyelinase generation of ceramide promotes clustering of nanoscale domains in supported bilayer membranes

The effects of ceramide incorporation in supported bilayers prepared from ternary lipid mixtures which have small nanoscale domains have been examined using atomic force and fluorescence microscopy. Both direct ceramide incorporation in vesicles used to prepare the supported bilayers and enzymatic hydrolysis of SM by sphingomyelinase were compared for membranes prepared from 5:5:1 DOPC/sphingomyelin/cholesterol mixtures. Both methods of ceramide incorporation resulted in enlargement of the initial small ordered domains. However, enzymatic ceramide generation led to a much more pronounced restructuring of the bilayer to give large clusters of domains with adjacent areas of a lower phase. The individual domains were heterogeneous with two distinct heights, the highest of which is assigned to a ceramide-rich phase which is hypothesized to occur via ceramide flip-flop to the lower leaflet with formation of a raised domain due to negative membrane curvature. A combination of AFM and fluorescence showed that the bilayer restructuring starts rapidly after enzyme addition, with formation of large clusters of domains at sites of high enzyme activity. The clustering of domains is accompanied by redistribution of fluid phase to the periphery of the domain clusters and there is a continued slow evolution of the bilayer over a period of an hour or more after the enzyme is removed. The relevance of the observed clustering of small nanoscale domains to the postulated coalescence of raft domains to form large signaling platforms is discussed.     

Sphingomyelinase generation of ceramide promotes clustering of nanoscale domains in supported bilayer membranes

The effects of ceramide incorporation in supported bilayers prepared from ternary lipid mixtures which have small nanoscale domains have been examined using atomic force and fluorescence microscopy. Both direct ceramide incorporation in vesicles used to prepare the supported bilayers and enzymatic hydrolysis of SM by sphingomyelinase were compared for membranes prepared from 5:5:1 DOPC/sphingomyelin/cholesterol mixtures. Both methods of ceramide incorporation resulted in enlargement of the initial small ordered domains. However, enzymatic ceramide generation led to a much more pronounced restructuring of the bilayer to give large clusters of domains with adjacent areas of a lower phase. The individual domains were heterogeneous with two distinct heights, the highest of which is assigned to a ceramide-rich phase which is hypothesized to occur via ceramide flip-flop to the lower leaflet with formation of a raised domain due to negative membrane curvature. A combination of AFM and fluorescence showed that the bilayer restructuring starts rapidly after enzyme addition, with formation of large clusters of domains at sites of high enzyme activity. The clustering of domains is accompanied by redistribution of fluid phase to the periphery of the domain clusters and there is a continued slow evolution of the bilayer over a period of an hour or more after the enzyme is removed. The relevance of the observed clustering of small nanoscale domains to the postulated coalescence of raft domains to form large signaling platforms is discussed.     

Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes

Sphingomyelin is an abundant component of eukaryotic membranes. A specific enzyme, sphingomyelinase can convert this lipid to ceramide, a central second messenger in cellular signaling for apoptosis (programmed cell death), differentiation, and senescence. We used microinjection and either Hoffman modulation contrast or fluorescence microscopy of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-sphingomyelin (C16:0-SM), and Bodipy-sphingomyelin as a fluorescent tracer (molar ratio 0.75:0.20:0.05, respectively) to observe changes in lipid lateral distribution and membrane morphology upon formation of ceramide. Notably, in addition to rapid domain formation (capping), vectorial budding of vesicles, i.e., endocytosis and shedding, can be induced by the asymmetrical sphingomyelinase-catalyzed generation of ceramide in either the outer or the inner leaflet, respectively, of giant phosphatidylcholine/sphingomyelin liposomes. These results are readily explained by 1) the lateral phase separation of ceramide enriched domains, 2) the area difference between the adjacent monolayers, 3) the negative spontaneous curvature, and 4) the augmented bending rigidity of the ceramide-containing domains, leading to membrane invagination and vesiculation of the bilayer.     

Differential effects of sphingomyelin hydrolysis and resynthesis on the activation of NF-kappa B in normal and SV40-transformed human fibroblasts

The precise role of ceramide in NF-kappaB signaling remains unclear. The recent observation of differential sphingomyelin synthase (SMS) activity in normal (low SMS) versus SV40-transformed (high SMS) WI38 human lung fibroblasts provides an opportunity to assess the involvement of ceramide and SMS in NF-kappaB activation. Treatment of normal WI38 fibroblasts with bacterial sphingomyelinase resulted in a 4-fold elevation of ceramide and blocked NF-kappaB activation by serum stimulation. Such inhibition was not observed in SV40-transformed fibroblasts. Under regular growth conditions, after sphingomyelinase was washed out, normal WI38 did not show SM re-synthesis nor NF-kappaB activation. In SV40-WI38, on the other hand, sphingomyelinase washout induced resynthesis of SM due to the action of SMS on ceramide generated at the plasma membrane. NF-kappaB activation correlated with SM resynthesis. This activation was abrogated by D609, which inhibited SM resynthesis but not the initial formation of ceramide. The differential activity of SMS may explain the effects of ceramide in NF-kappaB signaling: in the absence of significant SMS activity, ceramide inhibits NF-kappaB, whereas with high SMS, the conversion of the ceramide signal to a diacylglycerol signal by the action of SMS stimulates NF-kappaB. These results also suggest a role for SMS in regulating NF-kappaB.     

Ceramide stimulates a cytosolic protein phosphatase.

A sphingomyelin cycle has been identified whereby the action of certain extracellular agents results in reversible sphingomyelin hydrolysis and the concomitant generation of ceramide. Moreover, a cell-permeable ceramide, C2-ceramide (N-acetylsphingosine), is a potent modulator of cell proliferation and differentiation. We report herein that C2-ceramide, C6-ceramide, and natural ceramides activate a cytosolic serine/threonine protein phosphatase in a dose-dependent manner. Initial activation is observed at concentrations of ceramide as low as 0.1 microM with peak response occurring at 5-10 microM. However, other closely related sphingolipids, sphingosine and sphingomyelin, were largely inactive. Ceramide-stimulated phosphatase was inhibited by okadaic acid, an inhibitor of protein phosphatases, with an IC50 of 0.1-1 nM, depending on the concentration of ceramide. Ceramide-stimulated phosphatase was insensitive to Mg2+ and Mn2+ cations. Using sequential anion exchange chromatography, ceramide-stimulated phosphatase activity could be resolved from ceramide-nonresponsive phosphatases. The activity of partially purified enzyme was stimulated 3.5-fold by ceramide. The identification of a phosphatase as a molecular target for the action of ceramide defines a novel intracellular signaling pathway with potential roles in the regulation of cell proliferation and differentiation.     

Phase behavior and molecular interactions in mixtures of ceramide with dipalmitoylphosphatidylcholine.

In mixtures with dipalmitoylphosphatidylcholine, ceramide induces broadening of the calorimetric main phase transition that could be deconvoluted into at least three components: the first represents isothermal melting of a phosphatidylcholine-enriched phase; the second and third represent phases with increasing proportions of ceramide melting at progressively higher temperatures. The partial phase diagram (up to 40 mole % ceramide) indicates complete or partial gel-phase immiscibility, and complete gel- and liquid-phase miscibility depending on the ceramide content. Cluster distribution function analysis of each individual transition reveals decreased cooperativity and domain size with increased amounts of ceramide. Compared to individual lipids, mixed monolayers with dipalmitoylphosphatidylcholine show unchanged mean molecular areas or slight expansions at 24 degrees C with dipole potentials exhibiting hyperpolarization; by contrast, already at 27 degrees C the mean molecular areas become condensed and dipole potentials show little changes or are slightly depolarized. This suggests that favorable ceramide;-phosphatidylcholine dipolar matching in the liquid state can be one of the local determinants for close molecular interactions while unfavorable matching may explain lateral domain segregation of ceramide-enriched gel phases. The changes are detected at relatively low proportions of Cer (1;-12 mole %) which are comparable to variations of Cer levels in membranes of cultured cells undergoing functional responses mediated by the sphingomyelin signaling pathway.     

The role of sphingomyelin and sphingomyelin synthases in cell death,proliferation and migration—from cell and animal models to human disorders

Sphingomyelin constitutes membrane microdomains such as lipid raft, caveolae, and clathrin-coated pits and implicates in the regulation of trans-membrane signaling. On the other hand, sphingomyelin emerges as an important molecule to generate bioactive sphingolipids through ceramide. Sphingomyelin synthase is an enzyme that generates sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide. Although ceramide has a well-known role as a lipid mediator to regulate cell death and survival, the only known biological role of sphingomyelin regulated by sphingomyelin synthases was limited to being a source of bioactive lipids. Here, we describe the basic characters of sphingomyelin synthases and discuss additional roles for sphingomyelin and sphingomyelin synthase in biological functions including cell migration, apoptosis, autophagy, and cell survival/proliferation as well as in human disorders such as cancer and cardiovascular disorders. It is expected that a better understanding of the role of sphingomyelin regulated by sphingomyelin synthase will shed light on new mechanisms in cell biology, physiology and pathology. In the future, novel therapeutic procedures for currently incurable diseases could be developed through modifying the function of not only sphingolipids, such as sphingomyelin and ceramide, but also of their regulatory enzymes. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Functional reconstitution of sphingomyelin synthase in Chinese hamster ovary cell membranes.

Sphingomyelin synthase (phosphatidylcholine:ceramide phosphocholinetransferase) activity in the membranes of Chinese hamster ovary cells was found to be detectable with a fluorescent ceramide analog, containing a short acyl chain, as a substrate. We developed a method for the functional reconstitution of sphingomyelin synthase in detergent-treated membranes. Treatment of membranes with 1.5% octyl glucoside in the absence of exogenous phosphatidylcholine resulted in almost complete loss of sphingomyelin synthase activity, even after removal of the detergent by dialysis. In contrast, membranes treated with the detergent in the presence of exogenous phosphatidylcholine showed partial activity and, after dialysis of this mixture, enzyme activity was restored to almost the same level as the activity in dialyzed intact membranes. The effects of various lipids on enzyme activity in this reconstitution system suggested that L-alpha-phosphatidylcholine was the environmental lipid essential for the functional reconstitution of the enzyme. Furthermore, diacylglycerol was suggested to serve as an inhibitory regulator of sphingomyelin synthesis.     

The role of endogenous phosphatidylcholine and ceramide in the biosynthesis of sphingomyelin in mouse fibroblasts

The intracellular location of sphingomyelin formation via the cholinephosphotransferase reaction from both endogenous an exogenous phosphatidylcholine and ceramide substrates has been studied in the subcellular membrane fractions prepared from mouse fibroblasts. The enzyme was found to be located in both the plasma membrane and the Golgi fractions. Activity in the Golgi fraction was stimulated to a greater extent by the addition of exogenous ceramide than was the activity in the plasma membrane fraction. It is concluded that endogenous phosphatidylcholine is available to the cholinephosphotransferase at saturating concentration and, therefore, is not rate-limiting. In contrast, the very low concentration of endogenous ceramide seems to limit the reaction rate, necessitating supplementation with exogenous material Both endogenous substrates are shown to be utilized in an intramembranous rather than an intermembranous reaction. The capacity of the plasma membrane fraction to synthesize sphingomyelin from endogenous phosphatidylcholine and ceramide was found to be sufficiently high to account for the rate of net synthesis of plasma membrane-bound sphingomyelin observed in the logarithmically multiplying cell culture. In contrast, the Golgi fraction displayed only 26% of the expected capacity, but it was stimulated 6-fold by the addition of exogenous ceramide. These results demonstrate that the total cellular sphingomyelin of the mouse fibroblasts can be provided via the cholinephosphotransferase pathways and that the plasma membrane and the Golgi fraction are most probably the intracellular sites of sphingomyelin biosynthesis.     

Membrane microdomains: Role of ceramides in the maintenance of their structure and functions

Free-standing giant unilamellar vesicles were used to visualize the complex lateral heterogeneity, induced by ceramide in the membrane bilayer at micron scale using C₁₂-NBD-PC probe partitioning under the fluorescence microscope. Ceramide gel domains exist as leaf-like structures in glycerophospholipid/ceramide mixtures. Cholesterol readily increases ceramide miscibility with glycerophospholipids but cholesterol-ceramide interactions are not involved in the organization of the liquid-ordered phase as exemplified by sphingomyelin/cholesterol mixtures. Sphingomyelin stabilizes the gel phase and thus decreases ceramide miscibility in the presence of cholesterol. Gel/liquid-ordered/liquid-disordered phase coexistence was visualized in quaternary phosphatidylcholine/sphingomyelin/ceramide/cholesterol mixtures as occurrence of dark leaf-like and circular domains within a bright liquid phase. Sphingomyelin initiates specific ceramide-sphingomyelin interactions to form a highly ordered gel phase appearing at temperatures higher than pure ceramide gel phase in phosphatidylcholine/ceramide mixtures. Less sphingomyelin is engaged in formation of liquid-ordered phase leading to a shift in its formation to lower temperatures. Sphingomyelinase activity on substrate vesicles destroys micron L_o domains but induces the formation of a gel-like phase. The activation of phospholipase A₂ by ceramide on heterogeneous membranes was visualized. Changes in the phase state of the membrane bilayer initiates such morphological processes as membrane fragmentation, budding in and budding out was demonstrated.     

The formation of sphingomyelin from phosphatidylcholine in plasma membrane preparations from mouse fibroblasts

The enzymatic formation of radioactive sphingomyelin from [14C]choline-labeled phosphatidylcholine was demonstrated to reside exclusively in the plasma membrane fraction of mouse fibroblasts. This activity has several properties in common with the phosphatidylcholine ceramide phosphocholine transferase of mouse liver microsomes. The enzyme has little if any phospholipase C activity and isotope dilution experiments suggest that phosphatidylcholine is the substrate rather than it is converted to CDP choline, phosphocholine, free choline or glycerophosphocholine prior to the transfer reaction. The activity is stimulated by the addition of bovine serum albumin and MnCl2 to the incubation mixtures. The plasma membrane localization of the enzyme suggests that it may have a central role in the biosynthetic pathways for sphingomyelin in mouse fibroblasts.     

Solving the enigma: Mass spectrometry and small molecule probes to study sphingolipid function

Sphingolipids are highly bioactive lipids. Sphingolipid metabolism produces key membrane components (e.g. sphingomyelin) and a variety of signaling lipids with different biological functions (e.g. ceramide, sphingosine-1-phosphate). The coordinated activity of tens of different enzymes maintains proper levels and localization of these lipids with key roles in cellular processes. In this review, we highlight the signaling roles of sphingolipids in cell death and survival. We discuss recent findings on the role of specific sphingolipids during these processes, enabled by the use of lipidomics to study compositional and spatial regulation of these lipids and synthetic sphingolipid probes to study subcellular localization and interaction partners of sphingolipids to understand the function of these lipids.     

The role of sphingomyelin in regulating phase coexistence in complex lipid model membranes: competition between ceramide and cholesterol

The structure, thermotropic phase behavior, dynamic motion and order parameters of bilayer dispersions of egg phosphatidylcholine, egg sphingomyelin, egg ceramide and cholesterol have been determined. The coexistence of gel, liquid-ordered and liquid-disordered structure has been determined by peak fitting analysis of synchrotron X-ray powder patterns. Order parameters and extent of distribution of 16-doxyl-stearic acid spin probe between ordered and disordered environments has been estimated by ESR spectral simulation methods. The presence of ceramide in proportions up to 20 mol% in phosphatidylcholine is characterized by gel-fluid phase coexistence at temperatures up to 46 degrees C depending on the amount of ceramide. Cholesterol tends to destabilize the ceramide-rich domains formed in phosphatidylcholine while sphingomyelin, by formation of stable complexes with ceramide, tends to stabilize these domains. The stability of sphingomyelin-ceramide complexes is evident from the persistence of highly ordered structure probed by ESR spectroscopy and appearance of a sharp wide-angle X-ray reflection at temperatures higher than the gel-fluid transition of ceramide alone in egg phosphatidylcholine bilayers. The competition between ceramide and cholesterol for interaction with sphingomyelin is discussed in terms of control of lipid-mediated signaling pathways by sphingomyelinase and phospholipase A2.     

Sphingolipids of transplantable rat nephroma-RA

Contents of sphingolipids (ceramide, sphingomyelin, gangliosides) and the composition of their sphingoid bases were studied in the transplantable rat nephroma-RA and in rat kidneys. The content of sphingomyelin was about 1.3-fold decreased and the content of ceramide was about 1.4-fold increased in the nephroma compared to normal kidneys, and this correlated with a 1.4-fold increased activity of neutral sphingomyelinase; however, the activity of the acidic isoform of the enzyme was virtually unchanged. The content of gangliosides was also increased in the nephroma. Ceramide and sphingomyelin of the nephroma, in addition to sphingosine, contained a significant amount of sphinganine, although a considerable amount of the latter was also found in the renal ceramide. The ratio sphingosine/sphinganine in sphingomyelins changed from 65:1 in kidneys to 5:1 in the nephroma. Thus, the biosynthesis of sphingoid bases seems to be disturbed in the transplantable rat nephroma-RA compared to normal kidneys.     

Biosynthesis of sphingomyelin from erythro-ceramides and phosphatidylcholine by a microsomal cholinephosphotransferase

Mouse liver microsomes were shown to be active in the synthesis of sphingomyelin from ceramide and phosphatidylcholine in a reaction independent of CDPcholine. The conversion was not inhibited by calcium chelating reagents, and no evidence for the involvement of phospholipase C activity in the transformation could be adduced. Activity was also demonstrated in monkey liver and heart microsomes. Mouse brain microsomes produced a sphingomyelin analogue, tentatively identified as ceramide phosphorylethanolamine, but not sphingomyelin. Both [14C]ceramide and [G-14]phosphatidylethanolamine were precursors of the brain product, while phosphatidylcholine was inactive. Progress in the partial characterization of the liver enzyme is also described.     

The phosphorylcholine acceptor in the phosphatidylcholine:ceramide cholinephosphotransferase reaction. Is the enzyme a transferase or a hydrolase?

The cholinephosphotransferase reaction is shown to be catalyzed by an enzyme which has no hydrolytic activity and which is different from a phospholipase C type activity also present in these plasma membrane preparations. Diacylglycerols and sphingosine, at a concentration above 0.4 mM, are effective inhibitors of sphingomyelin formation in the presence of 0.3 mM free ceramide, the true acceptor in this reaction. Free sphingosine is not an acceptor for the cholinephosphate group, as the anticipated reaction product, sphingosylphosphocholine , could not be detected. Sphingosine inhibition may result from its structural similarity to the natural substrates of the reaction, ceramide and diacylglycerols. From the data obtained with cholesterol, triacylglycerols, acetylated ( triacetyl ) sphingosine and acetylated ceramides used as potential inhibitors of the reaction it is concluded that the free hydroxyl group at C1 of the sphingosine backbone or of the glycerol moiety of diacylglycerols and a non-polar residue consisting of an aliphatic chain were prerequisites for inhibitory activity. These results are discussed in terms of substrate specificity of the enzyme catalyzing the transfer reaction. Some of the factors influencing the regulation of the phosphatidylcholine/sphingomyelin ratio in the plasma membrane were related to the topography of sphingomyelin in the outer half-layer of the plasma membrane.     

Ceramide acyl chain length markedly influences miscibility with palmitoyl sphingomyelin in bilayer membranes

Ceramides are precursors of major sphingolipids and can be important cellular effectors. The biological effects of ceramides have been suggested to stem from their biophysical effects on membrane structure affecting the lateral and transbilayer organization of other membrane components. In this study we investigated the effect of acyl chain composition in ceramides (C4-C24:1) on their miscibility with N-palmitoyl-sphingomyelin (PSM) using differential scanning calorimetry. We found that short-chain (C4 and C8) ceramides induced phase separation and lowered the T _m and enthalpy of the PSM endotherm. We conclude that short-chain ceramides were more miscible in the fluid-phase than in the gel-phase PSM bilayers. Long-chain ceramides induced apparent heterogeneity in the bilayers. The main PSM endotherm decreased in cooperativity and enthalpy with increasing ceramide concentration. New ceramide-enriched components could be seen in the thermograms at all ceramide concentrations above X _Cer = 0.05. These broad components had higher T _m values than pure PSM. C24:1 ceramide exhibited complex behavior in the PSM bilayers. The miscibility of C24:1 ceramide with PSM at low (X _Cer = 0.05–0.10) concentrations was exceptionally good according to the cooperativity of the transition. At higher concentrations, multiple components were detected, which might have arisen from interdigitated gel-phases formed by this very asymmetric ceramide. The results of this study indicate that short-chain and long-chain ceramides have very different effects on the sphingomyelin bilayers. There also seems to be a correlation between their miscibility in binary systems and the effect of ceramides of different hydrophobic length on sphingomyelin-rich domains in multicomponent membranes.     

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.