Metabolomic profiling of sphingolipids in human glioma cell lines by liquid chromatography tandem mass spectrometry.

Sphingolipids participate in membrane structure and signaling in neuronal cells, and an emerging strategy for control of gliomas is to inhibit growth and/or induce apoptosis using ceramide and ceramide analogs. Nonetheless, some sphingolipids (ceramides and sphingosine) induce and others (sphingosine 1-phosphate) inhibit apoptosis; therefore, when testing putative anti-cancer agents, it is critical to obtain precise knowledge of the types and quantities of not only the test compounds, but also their effects on endogenous species. Combination of liquid chromatography and tandem mass spectrometry affords a "metabolomic" profile of all of the intermediates of ceramide biosynthesis (3-ketosphinganine, sphinganine and dihydroceramides) and the direct products of ceramide metabolism (sphingomyelins and monohexosylceramides as well as sphingosine and sphingosine 1-phosphate). This method has been applied to four human glioma cell lines (LN18, LN229, LN319 and T98G), and differences in the amounts and types of sphingolipids were found. For example, LN229 and LN319 have approximately twice the sphingosine 1-phosphate of LN18 and T98G; LN229 and LN319 have more monohexosylceramides than lactosylceramides, whereas the opposite is the case for LN18 and T98G; and the fatty acyl chain distributions of the sphingolipids differ among the cell lines. The ability to obtain this type of "metabolomic" profile allows studies of how anti-cancer agents (especially sphingolipids and sphingolipid analogs) affect the amounts of these bioactive species, and may lead to a better understanding of the abnormal phenotypes of gliomas.

Analysis of sphingosine 1-phosphate, ceramides, and other bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry.

The lipid backbones of sphingolipids and their metabolites are highly bioactive compounds that affect diverse cellular functions. The metabolites that have been most extensively studied with respect to their effects on cell behavior are ceramides, sphingosine (and other sphingoid bases), and sphingosine 1-phosphate. Additionally, there is interest in other naturally occurring species such as lysosphingolipids (sphingosine, phosphorylcholine, and psychosines) and N-methyl (di- and tri-methyl)-sphingosines. In many cases, studies of cell signaling mediated by these compounds have focused on a single category (such as ceramides or sphingosine 1-phosphate) because of the technical difficulty of more comprehensive analyses. One obstacle in such studies is that most of these compounds are metabolically interconvertable, so it is difficult to assign a conclusive relationship. In this article, we describe the analytical methods for extraction, identification, and quantitation of sphingolipids using state-of-the-art tandem mass spectrometry (MS/MS). Precursor ion scans are used to distinguish various species of sphingolipids in crude extracts by their unique molecular decomposition products. Specific headgroup, sphingoid base, and fatty acid chain combinations can be readily determined. Quantitation is achieved by multiple reaction monitoring (MRM) in conjunction with high-pressure liquid chromatography (HPLC). Compared to precursor ion scans alone, MRM experiments yield greater sensitivity and lower limits of detection by monitoring a specific precursor and product ion pair. This sensitivity facilitates detection of dozens of individual molecular species under optimal ion formation and decomposition conditions for each species, eliminating any ambiguity that may arise from differences in the kinetics of dissociation.

Modulation of intracellular beta-catenin localization and intestinal tumorigenesis in vivo and in vitro by sphingolipids.

Sphingolipid consumption suppresses colon carcinogenesis, but the specific genetic defect(s) that can be bypassed by these dietary components are not known. Colon tumors often have defect(s) in the adenomatous polyposis coli (APC)/beta-catenin regulatory system. Therefore, C57Bl/6J(Min/+) mice with a truncated APC gene product were fed diets supplemented with ceramide, sphingomyelin, glucosylceramide, lactosylceramide, and ganglioside G(D3) (a composition similar in amount and type to that of dairy products) to determine whether tumorigenesis caused by this category of genetic defect is suppressed. Sphingolipid feeding reduced the number of tumors in all regions of the intestine, and caused a marked redistribution of beta-catenin from a diffuse (cytosolic plus membrane) pattern to a more "normal" localization at mainly intercellular junctions between intestinal epithelial cells. The major digestion product of complex sphingolipids is sphingosine, and treatment of two human colon cancer cell lines in culture (SW480 and T84) with sphingosine reduced cytosolic and nuclear beta-catenin, inhibited growth, and induced cell death. Ceramides, particularly long-chain ceramides, also had effects. Thus, dietary sphingolipids, presumably via their digestion products, bypass or correct defect(s) in the APC/beta-catenin regulatory pathway. This may be at least one mechanism whereby dietary sphingolipids inhibit colon carcinogenesis, and might have implications for dietary intervention in human familial adenomatous polyposis and colon cancer.

Sphingolipid metabolism: roles in signal transduction and disruption by fumonisins.

Sphingolipids have important roles in membrane and lipoprotein structure and in cell regulation as second messengers for growth factors, differentiation factors, cytokines, and a growing list of agonists. Bioactive sphingolipids are formed both by the turnover of complex sphingolipids and as intermediates of sphingolipid biosynthesis. Usually, the amounts are highly regulated; however, by inhibiting ceramide synthase, fumonisins block the biosynthesis of complex sphingolipids and cause sphinganine (and sometimes sphingosine) to accumulate. Where the mechanism has been studied most thoroughly, the accumulation of sphingoid bases is a primary cause of the toxicity of fumonisin B (FB). Nonetheless, the full effects of fumonisins probably involve many biochemical events. The elevations in sphingoid bases also affect the amounts of other lipids, including the 1-phosphates and N-acetyl derivatives of sphinganine. Furthermore, the aminopentol backbone of FB1 (AP1) is both an inhibitor and a substrate for ceramide synthase, and the resultant N-palmitoyl-AP1 (PAP1) is an even more potent inhibitor of ceramide synthase (presumably as a product analog). PAP1 is 10 times more toxic than FB1 or AP1 for HT-29 cells in culture, and hence may play a role in the toxicity of nixtamalized fumonisins. All these processes--the effects of fumonisins on sphingolipid metabolism, the pathways altered by perturbation of sphingolipid metabolism, and the complex cellular behaviors regulated by sphingolipids--must be borne in mind when evaluating the pathologic effects of fumonisins.

Analysis of sphingomyelin, glucosylceramide, ceramide, sphingosine, and sphingosine 1-phosphate by tandem mass spectrometry.

Free sphingoid bases such as sphingosine, sphinganine, and the respective phosphorylated bases, as well as the complex sphingolipids ceramides, glucosylceramide, and sphingomyelin, all dissociate to form structurally distinctive product ions. For sphingomyelin these ions are characteristic of their phosphorylcholine headgroup and are observed at m/z 184. The other sphingolipids dissociate to form carbocations characteristic of their sphingoid base. For common mammalian sphingoid bases such as d18:1 or d18:0 these product ions are detected at m/z 264 or 266, respectively. However, changes in the sphingoid base chain length, degree of unsaturation, or other modifications may correspondingly result in a shift in m/z of [figure: see text] this product ion. Additionally, the kinetics that govern the formation of these product ions is affected by the presence of a delta 4 double bond. Thus, internal standards for each type of sphingoid base are required for quantitative data. Structurally distinctive product ions, when used with either precursor ion or constant neutral loss scans allow highly specific and sensitive methods for sphingolipid analysis. They serve to greatly reduce background chemical noise, and enhance detection of sphingolipids at very low concentrations. This occurs by allowing only those ions that dissociate to yield a specific product ion or neutral loss to be passed to the detector. Additionally, these scans reveal the exact combinations of headgroup, sphingoid base, and fatty acid in a complex mixture by mass. The free sphingoid bases and Cer readily decompose in the ion source, whereas GlcCer and SM do not. Finally, each individual sphingolipid species fragmented optimally at a different collision energy, precluding the use of either precursor ion or neutral loss scans for quantitation. Multiple reaction monitoring (MRM) experiments directly address the issues regarding accurate quantitation of sphingolipids that precursor ion and neutral loss scans cannot. In these experiments both ionization and dissociation parameters are optimized for each individual species. By detecting only specific precursor and product ion pairs instead of scanning wide m/z ranges maximum sensitivity is attained. Furthermore, relative ion abundance data are not biased with regard to instrumental parameters. At this point simple loop injections can be used with the MRM scanning methods developed to observe changes in sphingolipid type and quantity in crude extracts on a class-by-class basis. This, however, is labor intensive requiring multiple injections and multiple runs for each class in order to obtain a complete picture of all sphingolipids present. As an alternative [figure: see text] to loop injections, HPLC-MS/MS methods are being developed. In these methods sphingolipids are eluted by class, thus, each individually optimized MRM method can be used at specific times in an LC run. This provides a highly sensitive and accurate quantitation as well as a complete picture of all sphingolipids in a single run (Fig. 6). Additionally, this methodology is amenable to automation and can be used for high-throughput screening of multiple samples.

Structure determination of soybean and wheat glucosylceramides by tandem mass spectrometry.

Glucosylceramide (GluCer) is a major sphingolipid of plant tissue and, thus, abundant in nature and in dietary food sources. The lipid backbones of mammalian GluCer (sphingosine, d18:1(delta4), and ceramide) induce cell death (apoptosis) and inhibit colon carcinogenesis, it is critical to know the structures of GluCer present in plants as a first step toward understanding this potential link between diet and cancer. This study characterized the molecular species of GluCer from soybean and wheat by low-resolution, high-resolution and tandem mass spectrometry. Soybean GluCer was comprised primarily (>95%) of ceramide with 4,8-sphingadiene (d18:2(delta4,delta8)) and alpha-hydroxypalmitic acid (h16:0); the remainder had the same backbone with h18:0, h20:0, h22:0 and h24:0 fatty acids. Wheat GluCer had three major ceramide, d18:2(delta4,delta8) with h16:0, d18:1(delta8) with h16:0 and d18: 2(delta4,delta8) with h20:0, and smaller amounts of other homologs. These backbones differ from those of mammalian sphingolipids, which often have a delta4-double bond (but rarely a delta8-double bond), and have alpha-hydroxy fatty acids in only some cases. Previously unexplained fragmentations that were diagnostic for the type of sphingoid base backbone (i.e. by homolytic cleavage of the doubly allylic C-6-C-7 bond to yield a stable distonic allylic radical cation and an allylic radical neutral) were also identified. Hence this method should be useful in the identification of double bonds in sphingolipids, and structure-function relationships between sphingolipids and colon carcinogenesis.

Colonic cell proliferation and aberrant crypt foci formation are inhibited by dairy glycosphingolipids in 1, 2-dimethylhydrazine-treated CF1 mice.

Dietary sphingomyelin (SM) inhibits early stages of colon cancer (appearance of aberrant crypt foci, ACF) and decreases the proportion of adenocarcinomas vs. adenomas in 1,2-dimethylhydrazine (DMH)-treated CF1 mice. To elucidate the structural specificity of this inhibition, the effects of the other major sphingolipids in milk (glycosphingolipids) were determined. Glucosylceramide (GluCer), lactosylceramide (LacCer) and ganglioside G(D3) were fed individually to DMH-treated (six doses of 30 mg/kg body weight) female CF1 mice at 0.025 or 0.1 g/100 g of the diet for 4 wk. All reduced the number of ACF by > 40% (P < 0.001), which is comparable to the reduction by SM in earlier studies. Immunohistochemical analysis of the colons revealed that sphingolipid feeding also reduced proliferation, with the most profound effect (up to 80%; P < 0.001) in the upper half of the crypts. Since the bioactive backbones of the glycosphingolipids (i.e., ceramide and other metabolites) are the likely mediators of these effects, the susceptibility of these complex sphingolipids to digestion in the colon was examined by incubating 500 microgram of each sphingolipid with colonic segments from mice and analysis of substrate disappearance and product formation by tandem mass spectrometry. All of the sphingolipids (including SM) disappeared over time with a substantial portion appearing as ceramide. Partially hydrolyzed intermediates (such as GluCer from LacCer or G(D3)) were not detected, which suggests that the cleavage involves colonic (or microflora) endoglycosidases. In summary, consumption of dairy SM and glycosphingolipids suppresses colonic cell proliferation and ACF formation in DMH-treated mice; hence, many categories of sphingolipids affect these key events in colon carcinogenesis.

CD95(Fas/APO-1) signals ceramide generation independent of the effector stage of apoptosis.

Although numerous studies document caspase-independent ceramide generation preceding apoptosis upon environmental stress, the molecular ordering of ceramide generation during cytokine-induced apoptosis remains uncertain. Here, we show that CD95-induced ceramide elevation occurs during the initiation phase of apoptosis. We titrated down the amount of FADD transfected into HeLa and 293T cells until it was insufficient for apoptosis, although cycloheximide (CHX) still triggered the effector phase. Even in the absence of CHX, ceramide levels increased rapidly, peaking at 2.7 +/- 0.2-fold of control 8 h post-transfection. Dominant negative FADD failed to confer ceramide generation or CHX-mediated apoptosis. Ceramide generation induced by FADD was initiator caspase-dependent, being blocked by crmA. Limited pro-caspase 8 overexpression also increased ceramide levels 2.7 +/- 0.2-fold, yet failed, without CHX, to initiate apoptosis. Expression of membrane-targeted oligomerized CD-8 caspase 8 induced apoptosis without CHX, yet elevated ceramide only to a level equivalent to limited pro-caspase 8 transfection. Ceramide elevations were detected concurrently by diacylglycerol kinase and electrospray tandem mass spectrometry. These investigations provide evidence that ceramide generation is initiator caspase-dependent and occurs prior to commitment to the effector phase of apoptosis, definitively ordering ceramide as proximal in CD95 signaling.

Primary and secondary locations of charge sites in angiotensin II (M + 2H)2+ ions formed by electrospray ionization.

High-energy tandem mass spectrometry and molecular dynamics calculations are used to determine the locations of charge in metastably decomposing (M + 2H)2+ ions of human angiotensin II. Charge-separation reactions provide critical information regarding charge sites in multiple charged ions. The most probable kinetic energy released (Tm.p.) from these decompositions are obtained using kinetic energy release distributions (KERDs) in conjunction with MS/MS (MS2), MS/MS/MS (MS3), and MS/MS/MS/MS (MS4) experiments. The most abundant singly and doubly charged product ions arise from precursor ion structures in which one proton is located on the arginine (Arg) side chain and the other proton is located on a distal peptide backbone carbonyl oxygen. The MS3 KERD experiments show unequivocally that neither the N-terminal amine nor the aspartic acid (Asp) side chain are sites of protonation. In the gas phase, protonation of the less basic peptide backbone instead of the more proximal and basic histidine (His) side chain is favored as a result of reduced coulomb repulsion between the two charge sites. The singly and doubly charged product ions of lesser abundance arise from precursor ion structures in which one proton is located on the Arg side chain and the other on the His side chain. This is demonstrated in the MS3 and MS4 mass-analyzed ion kinetic energy spectrometry experiments. Interestingly, (b7" + OH)2+ product ions, like the (M + 2H)2+ ions of angiotensin II, are observed to have at least two different decomposing structures in which charge sites have a primary and secondary location.

Ceramide-beta-D-glucuronide: synthesis, digestion, and suppression of early markers of colon carcinogenesis.

Dietary sphingolipids inhibit chemically induced colon cancer in mice. The most likely mediators of this effect are the metabolites ceramide (Cer) and sphingosine, which induce growth arrest and apoptosis in transformed cells. Sphingolipids are digested in both the upper and the lower intestine; therefore, a more colon-specific method of delivery of sphingolipids might be useful. A Cer analogue with a D-glucuronic acid attached at the primary hydroxyl of N-palmitoyl-D-sphingosine (Cer-beta-glucuronide) was synthesized and evaluated as a substrate for Escherichia coli beta-glucuronidase and colonic digestion, as well as for suppression of early events in colon carcinogenesis in CFI mice treated with 1,2-dimethylhydrazine. Purified beta-glucuronidase (EC 3.2.1.31) and colonic segments (as a source of colonic enzymes and microflora) hydrolyzed Cer-beta-glucuronide to release Cer, as analyzed by tandem mass spectrometry. More than 75% of the Cer-beta-glucuronide was cleaved in an 8-h incubation with the colonic segments. When Cer-beta-glucuronide was administered for 4 weeks as 0.025% and 0.1% of the diet (AIN 76A) to 1,2-dimethylhydrazine-treated mice, there were significant reductions in colonic cell proliferation, as determined by in vivo BrdUrd incorporation, and in the appearance of aberrant crypt foci. The effect of dietary Cer-beta-glucuronide on aberrant crypt foci correlated significantly with the length of the colon, which suggests that Cer-beta-glucuronide was most effective when there was a larger compartment for digestion. Thus, synthetic sphingolipids that target the colon for the release of the bioactive backbones offer a promising approach to colon cancer prevention.

The importance of charge-separation reactions in tandem mass spectrometry of doubly protonated angiotensin II formed by electrospray ionization: Experimental considerations and structural implications.

The occurrence of charge-separation reactions in tandem mass spectrometry of doubly protonated angiotensin II is demonstrated by the use of mass-analyzed ion kinetic energy spectrometry (MIKES) and kinetic energy release distributions (KERDs). Linked scans at a constant B/E severely discriminate against product ions formed by charge-separation reactions. Although the products are significantly more abundant in MIKES experiments, instrumental discrimination still makes quantitation of relative product ion abundances highly inaccurate. The most probable KERs (T m. p.) and the average KERs (T ave.) of the reactions are determined from the KERDs, and these values are compared to the KERs determined from the peak widths at half-height (T 0. 5). The measurement of T 0. 5 is a poor approximation to T m. p. and T ave.. The T m. p. is used to calculate a most probable intercharge distance, which is compared to results from molecular dynamics calculations. The results provide evidence with regard to the mechanisms of fragmentation of multiply charged ions and the location of the charge site in relation to the decomposition reactions.

On the use of scans at a constant ratio of B/E for studying decompositions of peptide metal(II)-ion complexes formed by electrospray ionization.

The use of sector mass spectrometers to study metastable ion decompositions of peptide metal-ion complexes formed by electrospray ionization is discussed. Products that are formed by charge-separation reactions are characterized by large kinetic energy release distributions. This causes scans at a constant B/E to give incorrect product ion abundances and possibly incorrect mass assignments. Two instrumental methods exist that can be used either to detect the ions or to estimate relative ion abundances: a floated collision cell or mass-analyzed ion kinetic energy spectrometry (MIKES) scans. The floated collision cell, by virtue of an altered B/E scan law, however, discriminates against important metastable ion reactions that occur outside the cell. MIKES scans provide a clearer estimate of product ions that arise by metastable ion charge-separation reactions. Problems with pseudotandem (first field-free region) experiments are also discussed.