A review of traditional and current processing methods used to decrease the toxicity of the rhizome of Pinellia ternata in traditional Chinese medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Pinellia ternata (Thunb.) Breit, called Pinelliae Rhizoma (PR) and Banxia in Chinese, is a well-known traditional Chinese medicine (TCM) with the functions of "removing dampness-phlegm" and "downbear counterflow and check vomiting". PR has potential toxic effects that can be detoxified by Fuzhi processing (repeated processing using one or multiple adjuvants) with specific adjuvants. AIM OF THE STUDY: This paper aims to provide a summary of traditional and current processing methods used to detoxify PR. MATERIALS AND METHODS: The available references of the processing methods of PR from the classic books of Materia Medica, literature, online databases and masters or doctoral theses are collected and summarized. We also discussed the possible processing mechanisms of how we can achieve a safer and effective application of PR via these processing methods. RESULTS: PR cannot be administered orally before processing. PR contains nucleoside alkaloids, cerebrosides, fatty acids, lectin, polysaccharides, and calcium oxalate crystals. To date, although the active substances of PR are still unclear, the toxic components are almost completely clarified as needle-like calcium oxalate crystals (NCOCs) and lectin proteins. Furthermore, the toxic effects of PR include causing death in animals, inflammation, conjunctival irritation, pregnancy toxicity, teratogenicity, visceral toxicity, aphonia and vomiting. From ancient times to now, Fuzhi methods have remained the predominant method for PR processing, and the main adjuvants used are ginger juice, alum, licorice and lime. In addition, detoxification mechanisms are related to removing or damaging the NCOC and lectin in PR based on processing with adjuvants. Currently, Fuzhi processing has been greatly improved, and novel processing technologies with novel adjuvants have been used for PR processing. However, there are still some flaws in PR processing, which should be urgently solved in the future, and clarifying the characteristic bioactive compounds in PR corresponding to its function or effects is the most important step for PR processing. CONCLUSION: Our present paper reviewed the previous literature regarding all aspects of the processing of PR, and this paper will be helpful for achieving a safer and effective application of PR and its processed products and will also be beneficial for the further optimization of processing technology and clinical medication safety of PR.

Targeted analysis of ceramides and cerebrosides in yellow lupin seeds by reversed-phase liquid chromatography coupled to electrospray ionization and multistage mass spectrometry.

Ceramides (Cer) and cerebrosides are important sphingolipids (SL) involved in many biological processes. Herein, the SL content of yellow lupin seeds (Lupinus luteus) was determined by liquid-liquid extraction, mild alkaline hydrolysis (1 h at 37 °C) and reversed-phase liquid chromatography with negative electrospray ionization coupled to either an orbital-trap (Fourier-transformed, FT) or linear ion-trap (LIT) mass spectrometry (RPLC-ESI/MS). The chemical identity of SL including the sugar residues and sphingoid backbone (SB) was inferred by collision-induced dissociation multiple-stage mass spectrometry (MSn, n = 2,3). Up to 52 Cer and 47 cerebrosides were successfully recognized and quantified in sample extracts of L. luteus seeds also counting isobaric species. As reported for other vegetables, a hydroxylated SB was observed whereby the N-acyl chains showed a high occurrence of very-long-chain moieties; phytosphingosine and 4-hydroxy-8-sphingenine were the predominant SB paired mainly with oleic acid (C18:1), hydroxylated behenic acid (C22:0;1) and hydroxylated lignoceric acid (C24:0;1).

Cerebrosides and Steroids from the Edible Mushroom Meripilus giganteus with Antioxidant Potential.

The detailed chemical analysis of the methanol extract of Meripilus giganteus (Pers.) P. Karst. led to the isolation of two new cerebrosides, mericeramides A (1) and B (2) together with cerebroside B (3), ergosterol (4), 3ß-hydroxyergosta-7,22-diene (5), cerevisterol (6), 3ß-hydroxyergosta-6,8(14),22-triene (7), 3ß-O-glucopyranosyl-5,8-epidioxyergosta-6,22-diene (8) and (11E,13E)-9,10-dihydroxy-11,13-octadecadienoic acid (9). The structures of the compounds were determined on the basis of NMR and MS spectroscopic analysis. Mericeramide A (1) is the first representative of halogenated natural cerebrosides. The isolated fungal metabolites 1-9 were evaluated for their antioxidant activity using the oxygen radical absorbance capacity (ORAC) assay. Compounds 2, 5 and 9 proved to possess considerable antioxidant effects, with 2.50 ± 0.29, 4.94 ± 0.37 and 4.27 ± 0.05 mmol TE/g values, respectively. The result obtained gives a notable addition to the chemical and bioactivity profile of M. giganteus, highlighting the possible contribution of this species to a versatile and balanced diet.

Combining Salt Doping and Matrix Sublimation for High Spatial Resolution MALDI Imaging Mass Spectrometry of Neutral Lipids.

The combination of sodium salt doping of a tissue section along with the sublimation of the matrix 2,5-dihydrobenzoic acid (DHB) was found to be an effective coating for the simultaneous detection of neutral lipids and phospholipids using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry in positive ionization mode. Lithium, sodium, and potassium acetate were initially screened for their ability to cationize difficult to analyze neutral lipids such as cholesterol esters, cerebrosides, and triglycerides directly from a tissue section. The combination of sodium salt and DHB sublimation was found to be an effective cation/matrix combination for detection of neutral lipids. Further experimental optimizations revealed that sodium carbonate or sodium phosphate followed by DHB sublimation increases the signal intensity of the neutral lipids studied depending on the specific lipid family and tissue type by 10-fold to 140-fold compared with that of previously published methods. Application of sodium carbonate tissue doping and DHB sublimation resulted in crystal sizes ≤2 µm. We were thus able to image a mouse brain cerebellum at a high spatial resolution and detected 37 cerebrosides in a single run using a MALDI-TOF instrument. The combination of sodium doping and DHB sublimation offer a targeted and sensitive approach for the detection of neutral lipids that do not typically ionize well under normal MALDI conditions.

Mixing brain cerebrosides with brain ceramides, cholesterol and phospholipids.

The properties of bilayers composed of pure brain cerebroside (bCrb) or of binary mixtures of bCrb with brain ceramide, cholesterol, egg phosphatidylcholine or brain sphingomyelin have been studied using a combination of physical techniques. Pure bCrb exhibits a rather narrow gel-fluid transition centred at ≈65 °C, with a half-width at half-height T1/2 ≈ 3 °C. bCrb mixes well with both fluid and gel phospholipids and ceramide, and it rigidifies bilayers of egg phosphatidylcholine or brain sphingomyelin when the latter are in the fluid state. Cholesterol markedly widens the bCrb gel-fluid transition, while decreasing the associated transition enthalpy, in the manner of cholesterol mixtures with saturated phosphatidylcholines, or sphingomyelins. Laurdan and DPH fluorescence indicate the formation of fluid ordered phases in the bCrb:cholesterol mixtures. Macroscopic phase separation of more and less fluid domains is observed in giant unilamellar vesicles consisting of bCrb:egg phosphatidylcholine or bCrb:sphingomyelin. Crb capacity to induce bilayer permeabilization or transbilayer (flip-flop) lipid motion is much lower than those of ceramides. The mixtures explored here contained mostly bCrb concentrations >50 mol%, mimicking the situation of cell membranes in Gaucher's disease, or of the Crb-enriched microdomains proposed to exist in healthy cell plasma membranes.

[Cerebrosides isolated from Arisaema flavum].

Silica gel, Sephadex LH-20, and reverse phase (C-18) column chromatography were used for the research of chemical constituents occurred in Arisaema flavum(Forsk.) Schott. The structures were elucidated by comparison physico-chemical properties and NMR spectroscopic data with those of known compounds. Seventeen cerebrosides were identified as 1-O-ß-D-glucopyranosyl-(2S, 3R, 4E, 8E)-2-[(2'(R)-acetoxyoctadecanoyl)amido]-4, 8-octadecadiene-1, 3-diol (1), 2'-O-acetylsoyacerebroside I (2), 1-O-(ß-D-glucopyranosyl)-(2S, 3R, 4E, 13Z)-2-[(2'R)-2-hydroxytetradecanoylamino]-1, 3-dihydroxy-4, 13-docosadiene (3), (2S, 3R, 4E, 8E)1-(ß-D-glucopyranosyl)-3-hydroxy-2-[(R)-2'-hydroxyhexadecanoyl]amino-9-methyl-4, 8-heptadecadiene (4), (2S, 3R, 4E, 8E)1-(ß-D-glucopyranosyl)-3-hydroxy-2-[(R)-2'-hydroxyhexadecanoyl]amino-9-methyl-4, 8-octadecadiene (5), (2S, 3R, 4E, 8E)1-(ß-D-glucopyranosyl)-3-hydroxy-2-[(R)-2'-hydroxypalmitoyl]amino-9-methyl-4, 8-octadecadiene (6), (2S, 3R, 4E, 8E)1-(ß-D-glucopyranosyl)-3-hydroxy-2-[(R)-2'-hydroxyoctadecanoyl]amino-9-methyl-4, 8-octadecadiene (7), 1-O-(ß-D-glucopyranosyl)-(2S, 3R, 4E, 8E)-2-[(R)-2'-hydroxytetradecanoylamino]-4, 8-octadecadiene-1, 3-diol (8), 1-O-(ß-D-glucopyranosyl)-(2S, 3R, 4E, 8E)-2-[(R)-2'-hydroxypentadecanoylamino]-4, 8-octadecadiene-1, 3-diol (9), 1-O-(ß-D-glucopyranosyl)-(2S, 3R, 4E, 8E)-2-[(R)-2'-hydroxyhexadecanoylamino]-4, 8-octadecadiene-1, 3-diol (10), 1-O-(ß-D-glucopyranosyl)-(2S, 3R, 4E, 8Z)-2-[(R)-2'-hydroxyhexadecanoylamino]-4, 8-octadecadiene-1, 3-diol (11), 1-O-(ß-D-glucopyranosyl)-(2S, 3R, 4E, 8E)-2-[(R)-2'-hydroxyoctadecanoylamino]-1, 3-hydroxy-4, 8-octadecadiene (12), 1-O-(ß-D-glucopyranosyl)-(2S, 3R, 4E)-2-[(R)-2'-hydroxytetracosanoylamino]-1, 3-hydroxy-4-hexadecane (13), 1-O-(ß-D-glucopyranosyl)-(2S, 3R, 4R, 8Z)-2N-[(2'R)-2'-hydroxytetracosanoyl]-8-(Z)-octadecene-1, 3, 4-triol (14), 1-O-(ß-D-glucopyranosyl)-(2S, 3S, 4E, 8E)-2N-[(2'R)-2'-hydroxyhexadecanoyl]-4-(E), 8-(Z)-octadecadiene-1, 3-diol (15), typhoniside A (16), and 1-O-ß-D-glucopyranosyl-(2S, 3R, 8E)-2-[(2'R)-2-hydroxypalmitoylamino]-8-octadecene-1, 3-diol (17). Compounds 1 and 2 were isolated from the plant for the first time, while the remained compounds were isolated from the genus Arisaema for the first time.

Δ10(E)-Sphingolipid Desaturase Involved in Fusaruside Mycosynthesis and Stress Adaptation in Fusarium graminearum.

Sphingolipids are biologically important and structurally distinct cell membrane components. Fusaruside (1) is a 10,11-unsaturated immunosuppressive fungal sphingolipid with medical potentials for treating liver injury and colitis, but its poor natural abundance bottlenecks its druggability. Here, fusaruside is clarified biosynthetically, and its efficacy-related 10,11-double bond can be generated under the regioselective catalysis of an unprecedented Δ10(E)-sphingolipid desaturase (Δ10(E)-SD). Δ10(E)-SD shares 17.7% amino acid sequence similarity with a C9-unmethylated Δ10-sphingolipid desaturase derived from a marine diatom, and 55.7% with Δ8(E)-SD from Fusarium graminearum. Heterologous expression of Δ10(E)-SD in Pichia pastoris has been established to facilitate a reliable generation of 1 through the Δ10(E)-SD catalyzed desaturation of cerebroside B (2), an abundant fungal sphingolipid. Site directed mutageneses show that the conserved histidines of Δ10(E)-SD are essential for the 10,11-desaturation catalysis, which is also preconditioned by the C9-methylation of the substrate. Moreover, Δ10(E)-SD confers improved survival and faster growth to fungal strains at low temperature and high salinity, in parallel with to higher contents of 1 in the mycelia. Collectively, the investigation describes a new Δ10(E)-sphingolipid desaturase with its heterologous expression fundamentalizing a biotechnological supply of 1, and eases the follow-up clarification of the immunosuppression and stress-tolerance mechanism.

Cerebrosides from a Far-Eastern Glass Sponge Aulosaccus sp.

Nine new cerebrosides 1a-d, 2a, 2b, 3a-c were found in the extract of a Far-Eastern glass sponge Aulosaccus sp. (class Hexactinellida). These ß-D-glucopyranosyl-(1 → 1)-ceramides contain sphingoid bases (2S,3S,4R,11Z)-2-aminoeicos-11-ene-1,3,4-triol (in 1a-d), (2S,3S,4R,13Z)-2-aminoeicos-13-ene-1,3,4-triol (in 2a, b) and (2S,3S,4R,13S*,14R*)-2-amino-13,14-methylene-eicosane-1,3,4-triol (in 3a-c), which are N-acylated by (2R,15Z)-2-hydroxydocos-15-enoic (in 1a, 2a, 3a), (2R,16Z)-2-hydroxytricos-16-enoic (in 1b, 2b, 3b), (2R,17Z)-2-hydroxytetracos-17-enoic (in 1d) and (2R)-2-hydroxydocosanoic (in 1c, 3c) acids. The monoenoic and cyclopropane-containing sphingoid bases of compounds 1a-d, 2a, 2b, 3a-c have not been found previously in any sphingolipids. The structures of the cerebrosides were elucidated on the basis of (1)H-, (13)C-NMR spectroscopy, mass spectrometry, optical rotation data and chemical transformations. A simplified method for the assignment of the absolute configuration of 2-hydroxy fatty acids by GC analysis of their (2R)- and (2S)-oct-2-yl esters was proposed.

High throughput analysis of cerebrosides from the sea cucumber Pearsonothria graeffei by liquid chromatography-quadrupole-time-of-flight mass spectrometry.

Liquid chromatography-mass spectrometry was one of the most powerful methods for identification and detection of chemical structures of lipids. In this study, the cerebrosides molecular species from the sea cucumber Pearsonothria graeffei (P. graeffei) were high throughput identified by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS). Cerebrosides were separated and purified by solid-phase extraction with chloroform-methanol solution. Precursor ion scan spectra and product ion scan spectra were obtained through auto MS/MS analysis in the positive scan. Cerebroside molecules were selected according to the neutral loss fragments of 180 Da, and then the structures were identified according to pairs of specific products of sphingoid bases and their precursor ions. Eighty-nine cerebrosides molecular species were identified, large amounts of d17:1-C22:0 h, t17:0-C24:1h, d17:1-C24:1h, d17:1-C23:0 h, d17:1-C22:0 and d17:1-C23:0 were present which have hardly found in mammal. There were 13 classes of long-chain base (LCB), and the ratio of phytosphingosines and sphingosines was roughly 1:9, in which two of the most common LCBs were d17:1 and d18:1. The carbon numbers of fatty acids (FAs) were mainly 18~24, while 24 carbon fatty acids were predominant. The ratio of saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) was about 2:3, and the percentage of hydroxy fatty acid (HFA) was over 60%. The ratio of non-hydroxylated fatty acid (NFA)/HFA was also approximately 2:3. LC-Q-TOF-MS analysis should be useful for the structure determination of diverse cerebrosides molecular species. Meanwhile, this method provided a basis for structure-activity relationship studies and functional food development of the sea cucumber P. graeffei as well.

[High throughput analysis of cerebroside molecular species from sea cucumber Parastichopus californicus by liquid chromatography-quadrupole-time-of-flight mass spectrometry].

Cerebrosides from sea cucumber Parastichopus californicus were identified by using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOF MS/ MS). The samples were extracted with chloroform-methanol (2: 1, v/v) solution and purified by a SPE cartridge. In positive ion mode electrospray ionization (ESI), the precursor ion scan mass spectra and product ion scan mass spectra were obtained through the automatic MS/MS mode. Cerebroside molecules were selected according to the neutral loss fragments of 180 Da, and then were identified according to long-chain base (LCB) fragments and fatty acid (FA) fragments. One hundred and twenty-three cerebroside molecular species were identified. There are 18 species of LCB, and the relative content ratio of phytosphingosines and sphingosines is 1: 2. The carbon numbers of fatty acids are mainly 18 - 25, of which 24 carbon fatty acids are predominant. The relative content ratio of saturated fatty acids and monounsaturated fatty acid is about 1: 3, and the presence of 2-hydroxy fatty acids is about 58.62%. LC-Q-TOF MS/MS method is sensitive, accurate and simple. At the same time, this study provided a theoretical basis for structure-activity relationship studies and functional food development of Parastichopus californicus as well.

Lipid composition of the stratum corneum and cutaneous water loss in birds along an aridity gradient.

Intercellular and covalently bound lipids within the stratum corneum (SC), the outermost layer of the epidermis, are the primary barrier to cutaneous water loss (CWL) in birds. We compared CWL and intercellular SC lipid composition in 20 species of birds from desert and mesic environments. Furthermore, we compared covalently bound lipids with CWL and intercellular lipids in the lark family (Alaudidae). We found that CWL increases in birds from more mesic environments, and this increase was related to changes in intercellular SC lipid composition. The most consistent pattern that emerged was a decrease in the relative amount of cerebrosides as CWL increased, a pattern that is counterintuitive based on studies of mammals with Gaucher disease. Although covalently bound lipids in larks did not correlate with CWL, we found that covalently bound cerebrosides correlated positively with intercellular cerebrosides and intercellular cholesterol ester, and intercellular cerebrosides correlated positively with covalently bound free fatty acids. Our results led us to propose a new model for the organization of lipids in the avian SC, in which the sugar moieties of cerebrosides lie outside of intercellular lipid layers, where they may interdigitate with adjacent intercellular cerebrosides or with covalently bound cerebrosides.

Severe alterations in lipid composition of frontal cortex lipid rafts from Parkinson's disease and incidental Parkinson's disease.

Lipid rafts are cholesterol- and sphingomyelin-enriched microdomains that provide a highly saturated and viscous physicochemical microenvironment to promote protein-lipid and protein-protein interactions. We purified lipid rafts from human frontal cortex from normal, early motor stages of Parkinson's disease (PD) and incidental Parkinson's disease (iPD) subjects and analyzed their lipid composition. We observed that lipid rafts from PD and iPD cortices exhibit dramatic reductions in their contents of n-3 and n-6 long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (22:6-n3) and arachidonic acid (20:4n-6). Also, saturated fatty acids (16:0 and 18:0) were significantly higher than in control brains. Paralleling these findings, unsaturation and peroxidability indices were considerably reduced in PD and iPD lipid rafts. Lipid classes were also affected in PD and iPD lipid rafts. Thus, phosphatidylserine and phosphatidylinositol were increased in PD and iPD, whereas cerebrosides and sulfatides and plasmalogen levels were considerably diminished. Our data pinpoint a dramatic increase in lipid raft order due to the aberrant biochemical structure in PD and iPD and indicate that these abnormalities of lipid rafts in the frontal cortex occur at early stages of PD pathology. The findings correlate with abnormal lipid raft signaling and cognitive decline observed during the development of these neurodegenerative disorders.

Qualitative on-line profiling of ceramides and cerebrosides by high performance liquid chromatography coupled with electrospray ionization ion trap tandem mass spectrometry: the case of Dracontium loretense.

Ceramides and cerebrosides are key compounds in the metabolism of sphingolipids. Produced in response to a variety of apoptotic stimuli, these metabolites mediate either mitogenic or apoptotic responses, depending on cell type and nature of stimulus. Novel strategies using these selective targets for a therapeutic intervention, e.g. in cancer, cardiovascular and neurodegenerative diseases, and HIV, have been developed, along with anticancer approaches using controlled delivery of exogenous natural ceramides from ceramide-based liposomes. Thus, great is the need to find selective and sensitive analytical methods allowing a prompt detection of ceramides and cerebrosides in natural matrices. Here we report an analytical study carried out on the Amazonian plant Dracontium loretense, resulted in a preliminary analysis a rich source of this class of natural compounds. A handy, selective, and sensitive methodology based on high-performance liquid chromatography coupled to electrospray negative ionization multistage ion trap mass spectrometry (HPLC-ESI/ITMS(n)) was developed. Analysis of fingerprint multistage mass spectra allowed the rapid identification of 3 major long-chain bases and their exact pairing with 11 different fatty acids and with carbohydrate headgroups. Thus, the structures of 21 ceramide and cerebroside species, among which 7 molecules never reported before, were unambiguously assigned. Results obtained in this study demonstrated that this analytical approach could provide a reliable and sensitive method to obtain the qualitative on-line profiling of ceramides and cerebrosides in new medicinal plant matrices.

Glycosylceramides obtain from the starfish Asterias amurensis Lütken.

Complex lipids in the starfish Asterias amurensis were characterized and the influence of sphingoid bases on human colon carcinoma Caco-2 cells was also investigated. Lipid content of gonad and viscera were 3.3% and 6.8%, respectively, in wet basis. The main lipid class in gonad was ceramide monohexoside (CMH) while triglyceride (TG) was predominant in the viscera. The most abundant fatty acid in the polar lipid was eicosapentaenoic acid (EPA, C20:5n-3), with the gonad and viscera samples having the highest proportion of 41.5% and 32.7%, respectively, of total fatty acids. Starfish internal organ contained enormous amount (0.7% in wet base) of glycosylceramide. Sphingoid bases of the glycosylceramide were mainly consisted of d22:2, d22:1 and d18:3. This sphingoid base exerted an apoptotic activity on Caco-2 cells. Thus, starfish could be used as a potential source of precious and useful complex lipids.

Analysis of sphingolipid classes and their contents in meals.

Sphingolipids have attracted attention as physiologically functional lipids. We determined their class and content in Japanese meals that had been prepared by a nutritionist, mainly by using HPLC-ELSD. In all 12 meals tested, cerebroside and/or sphingomyelin were generally detected as the major sphingolipids. The total amounts of sphingolipids in typical high- and low-calorie meal samples over 2 days were 292 and 128 mg/day, and 81 and 45 mg/day, respectively.

Studies of oxidative stress mechanisms using a morphine / ascorbate animal model and novel N-stearoyl cerebroside and laurate sensors.

The field of oxidative stress, free radicals, cellular defense and antioxidants is a burgeoning field of research. An important biomarker of oxidative stress is ascorbate and alterations in ascorbate have been shown to be a reliable measure of oxidative stress mechanisms. The purpose of this pharmacological study was to assess changes in ascorbate in a morphine/ascorbate animal model using novel sensors which selectively detect electrochemical signals for ascorbate, dopamine (DA) and serotonin (5-HT). Studies were also performed to show reversal of morphine-induced effects by the opioid antagonist, naloxone. In vivo studies were modeled after (Enrico et al. 1997, 1998) in which the oxidative biomarker, ascorbate, was reported to compensate for free radicals produced by morphine-induced increases in DA and 5-HT. In vivo studies consisted of inserting the Laurate sensor in ventrolateral nucleus accumbens (vlNAcc), in anesthetized male, Sprague-Dawley rats. In separate studies, laboratory rats were injected with (1) ascorbate, (5-35 mg/kg, ip) or (2) dehydroascorbate (DHA) (20-100 mg/kg, ip). In another study, (3) morphine sulfate (10-20 mg/kg, sc) was injected followed by a single injection of naloxone (5 mg/kg, ip) in the same animal. Results showed that in vlNAcc, (1) neither ascorbate nor DHA injections produced ascorbate release, (2) morphine significantly increased DA and 5-HT release, but did not alter ascorbate release, and (3) naloxone significantly reversed the increased DA and 5-HT release produced by morphine. Moreover, the sensors, N-stearoyl cerebroside and laurate were studied in vitro, in separate studies, in order to assess selective and separate electrochemical detection of ascorbate, DA and 5-HT, neuromolecules involved in oxidative stress mechanisms. In vitro studies consisted of pretreatment of each sensor with a solution of phosphotidylethanolamine (PEA) and bovine serum albumin (BSA) which simulates the lipid/protein composition of brain. Each new sensor was tested for stability, sensitivity and selectivity by pipetting graduated increases in concentration of ascorbate, DA and 5-HT into an electrochemical cell containing saline/phosphate buffer. Multiple and repetitive images of electrochemical signals from ascorbate, DA and 5-HT were recorded. Results showed that both sensors produced three well-defined cathodic, selective and separate electrochemical signals for ascorbate, DA and 5-HT at characteristic oxidation potentials. Dopamine and 5-HT were detected at nM concentrations while ascorbate was detected at microM concentrations. In summary, the data show that very low concentrations of ascorbate occurred in vlNAcc since novel sensors detected ascorbate at high concentrations in vitro. The data indicate that little or no change in oxidative stress mechanisms occurred in vlNAcc after morphine or naloxone administration since the oxidative biomarker, ascorbate, was not signifi cantly altered. Thus, oxidative stress mechanisms and novel N-stearoyl cerebroside and laurate sensors, which selectively detect and separate neuromolecules involved in these mechanisms, may be potentially clinically relevant.

MALDI-ion mobility-TOFMS imaging of lipids in rat brain tissue.

While maintaining anatomical integrity, matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) has allowed researchers to directly probe tissue, map the distribution of analytes and elucidate molecular structure with minimal preparation. MALDI-ion mobility (IM)-orthogonal time-of-flight mass spectrometry (oTOFMS) provides an advantage by initially separating different classes of biomolecules such as lipids, peptides, and nucleotides by their IM drift times prior to mass analysis. In the present work the distribution of phosphatidlycholine and cerebroside species was mapped from 16 microm thick coronal rat brain sections using MALDI-IM-oTOFMS. Furthermore, the use of gold nanoparticles as a matrix enables detection of cerebrosides, which although highly concentrated in brain tissue, are not easily observed as positive ions because of intense signals from lipids such as phosphatidlycholines and sphingomyelins.

Analysis of lipids using 2,4,6-trihydroxyacetophenone as a matrix for MALDI mass spectrometry.

Lipids exhibit a broad range of chemical properties that make their analysis quite demanding. Today, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) represents a versatile tool in the field of lipid analysis, also offering the possibility for molecular structural identification using novel MALDI tandem time-of-flight (TOF/TOF) instrumentation. In this study, we evaluated 2,4,6-trihydroxyacetophenone (THAP) for the analysis of various lipid classes including neutral storage lipids (triacylglycerols), polar membrane lipids (glycerophospho- and sphingolipids), and glycosphingolipids. THAP proved to be a versatile matrix for the routine analysis of various lipids from biological samples ("lipidomics"). A sample preparation methodology was established using selective alkali salt doping for subsequent MS/MS experiments. Sodiated and lithiated molecules provided superior structural information on lipids (i.e., acyl group identification); thus, following this approach, both selective peak detection with high sensitivity and more reliable structural information were obtained simultaneously.

Sequential pressurized liquid extraction to determine brain-originating fatty acids in meat products as markers in bovine spongiform encephalopathy risk assessment studies.

A new approach using sequential pressurized liquid extraction described recently [J. Poerschmann, R. Carlson, J. Chromatogr. A, 1127 (2006) 18-25] was applied to determine lipid markers originating from central nervous system (CNS) tissue of cows in heat-processed sausages. These studies are very important in quality control as well as risk assessment studies in the face of the bovine spongiform encephalopathy (BSE) crisis. Diagnostic CNS lipid markers, which should not be present in meat products without CNS addition, were recognized on complete transesterification as polar 2-hydroxy-fatty acids (2OH-24:0, 2OH-24:1, 2OH-22:0, 2OH-18:0, shorthand designation) as well as odd-numbered non-branched fatty acids beyond C(22). An array of other fatty acids including lignoceric acid (24:0), nervonic acid (24:1), arachidonic acid (20:4), and polyunsaturated nC(22)-surrogates are strongly related to CNS lipids, but occur as traces in meat products without CNS addition as well, thus reducing their value as diagnostic markers. Samples including meat products without CNS addition, meat with 3% CNS addition, as well as pure CNS homogenates, were subjected to sequential PLE (pressurized liquid extraction) consisting of two steps: n-hexane/acetone 9:1 (v/v) extraction at 50 degrees C to remove neutral lipids, followed by chloroform/methanol 1:4 (v/v) extraction at 110 degrees C to isolate polar CNS lipids (two 10 min PLE cycles each). To enhance the fractionation efficiency, cyanopropyl modified silica as well as chemically not modified silica sorbent was used at the outlet of the PLE cartridge to retard polar lipids in the first extraction step. This method proved superior to widely distributed exhaustive lipid extraction followed by solid-phase extraction (SPE) using silica regarding lipid recoveries and clear-cut boundaries between lipid classes. Methodological studies showed that the alcoholysis using trimethylchlorosilane/methanol (1:9, v/v) is an excellent method for the complete transesterification of lipids and quantitative formation of methyl esters.