Structural Determination of Lysosphingomyelin-509 and Discovery of Novel Class Lipids from Patients with Niemann-Pick Disease Type C.

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder caused by the mutation of cholesterol-transporting proteins. In addition, early treatment is important for good prognosis of this disease because of the progressive neurodegeneration. However, the diagnosis of this disease is difficult due to a variety of clinical spectrum. Lysosphingomyelin-509, which is one of the most useful biomarkers for NPC, was applied for the rapid and easy detection of NPC. The fact that its chemical structure was unknown until recently implicates the unrevealed pathophysiology and molecular mechanisms of NPC. In this study, we aimed to elucidate the structure of lysosphingomyelin-509 by various mass spectrometric techniques. As our identification strategy, we adopted analytical and organic chemistry approaches to the serum of patients with NPC. Chemical derivatization and hydrogen abstraction dissociation-tandem mass spectrometry were used for the determination of function groups and partial structure, respectively. As a result, we revealed the exact structure of lysosphingomyelin-509 as N-acylated and O-phosphocholine adducted serine. Additionally, we found that a group of metabolites with N-acyl groups were increased considerably in the serum/plasma of patients with NPC as compared to that of other groups using targeted lipidomics analysis. Our techniques were useful for the identification of lysosphingomyelin-509.

Developmental Changes for the Hemolymph Metabolome of Silkworm (Bombyx mori L.).

Silkworm (Bombyx mori) is a lepidopteran-holometabolic model organism. To understand its developmental biochemistry, we characterized the larval hemolymph metabonome from the third instar to prepupa stage using (1)H NMR spectroscopy whilst hemolymph fatty acid composition using GC-FID/MS. We unambiguously assigned more than 60 metabolites, among which tyrosine-o-ß-glucuronide, mesaconate, homocarnosine, and picolinate were reported for the first time from the silkworm hemolymph. Phosphorylcholine was the most abundant metabolite in all developmental stages with exception for the periods before the third and fourth molting. We also found obvious developmental dependence for the hemolymph metabonome involving multiple pathways including protein biosyntheses, glycolysis, TCA cycle, the metabolisms of choline amino acids, fatty acids, purines, and pyrimidines. Most hemolymph amino acids had two elevations during the feeding period of the fourth instar and prepupa stage. Trehalose was the major blood sugar before day 8 of the fifth instar, whereas glucose became the major blood sugar after spinning. C16:0, C18:0 and its unsaturated forms were dominant fatty acids in hemolymph. The developmental changes of hemolymph metabonome were associated with dietary nutrient intakes, biosyntheses of cell membrane, pigments, proteins, and energy metabolism. These findings offered essential biochemistry information in terms of the dynamic metabolic changes during silkworm development.

Lyso-sphingomyelin is elevated in dried blood spots of Niemann-Pick B patients.

Niemann-Pick disease type B (NPD-B) is caused by a partial deficiency of acid sphingomyelinase activity and results in the accumulation of lysosomal sphingomyelin (SPM) predominantly in macrophages. Notably, SPM is not significantly elevated in the plasma, whole blood, or urine of NPD-B patients. Here, we show that the de-acylated form of sphingomyelin, lyso-SPM, is elevated approximately 5-fold in dried blood spots (DBS) from NPD-B patients and has no overlap with normal controls, making it a potentially useful biomarker.

Absolute quantification of choline-related biomarkers in breast cancer biopsies by liquid chromatography electrospray ionization mass spectrometry.

It has been repeatedly demonstrated that choline metabolism is altered in a wide variety of cancers. In breast tumours, the choline metabolite profile is characterized by an elevation of phosphocholine and total choline-compounds. This pattern is increasingly being exploited as biomarker in cancer diagnosis. The majority of in vitro metabolomics studies, for biomarkers quantification in cell cultures or tissues, entail proton NMR spectroscopy. Although many "targeted" approaches have been proposed to quantify metabolites from standard one-dimensional (1D) NMR experiments, the task is often made difficult by the high degree of overlap characterizing 1H NMR spectra of biological samples. Here we present an optimized protocol for tissue extraction and absolute quantification of choline, phosphocholine and glycerophosphocholine by means of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The selected chromatographic separation system with a HILIC (hydrophilic interaction chromatography) amide column effectively separates free choline and its phopshorylated derivatives, contrary to failure observed using standard reversed-phase chromatography. The metabolite absolute quantification is based on external calibration with commercial standards, and is validated by a parallel 1D proton NMR analysis. The LC-MS/NMR analysis is applied to three breast carcinoma specimens obtained by surgical excision, each one accompanied by a control tissue sample taken outside the tumor margin. The metabolite concentrations measured are in good agreement with previous results on metabolic profile changes of breast cancer. Each of the three cancerous biopsies, when compared with the control tissue, exhibit a highly increased levels phosphocholine, total choline and phosphocholine/glycerophosphocholine ratio.

Metabolic signature of electrosurgical liver dissection.

BACKGROUND AND AIMS: High frequency electrosurgery has a key role in the broadening application of liver surgery. Its molecular signature, i.e. the metabolites evolving from electrocauterization which may inhibit hepatic wound healing, have not been systematically studied. METHODS: Human liver samples were thus obtained during surgery before and after electrosurgical dissection and subjected to a two-stage metabolomic screening experiment (discovery sample: N = 18, replication sample: N = 20) using gas chromatography/mass spectrometry. RESULTS: In a set of 208 chemically defined metabolites, electrosurgical dissection lead to a distinct metabolic signature resulting in a separation in the first two dimensions of a principal components analysis. Six metabolites including glycolic acid, azelaic acid, 2-n-pentylfuran, dihydroactinidiolide, 2-butenal and n-pentanal were consistently increased after electrosurgery meeting the discovery (p<2.0 × 10(-4)) and the replication thresholds (p<3.5 × 10(-3)). Azelaic acid, a lipid peroxidation product from the fragmentation of abundant sn-2 linoleoyl residues, was most abundant and increased 8.1-fold after electrosurgical liver dissection (preplication = 1.6 × 10(-4)). The corresponding phospholipid hexadecyl azelaoyl glycerophosphocholine inhibited wound healing and tissue remodelling in scratch- and proliferation assays of hepatic stellate cells and cholangiocytes, and caused apoptosis dose-dependently in vitro, which may explain in part the tissue damage due to electrosurgery. CONCLUSION: Hepatic electrosurgery generates a metabolic signature with characteristic lipid peroxidation products. Among these, azelaic acid shows a dose-dependent toxicity in liver cells and inhibits wound healing. These observations potentially pave the way for pharmacological intervention prior liver surgery to modify the metabolic response and prevent postoperative complications.

Sphingosylphosphorylcholine as a novel calmodulin inhibitor.

S1P (sphingosine 1-phosphate) and SPC (sphingosylphosphorylcholine) have been recently recognized as important mediators of cell signalling, regulating basic cellular processes such as growth,differentiation, apoptosis, motility and Ca2+ homoeostasis.Interestingly, they can also act as first and second messengers. Although their activation of cell-surface G-protein-coupled receptors has been studied extensively, not much is known about heir intracellular mechanism of action, and their target proteins are yet to be identified. We hypothesized that these sphingolipids might bind to CaM (calmodulin), the ubiquitous intracellular Ca2+sensor. Binding assays utilizing intrinsic tyrosine fluorescence of the protein, dansyl-labelled CaM and surface plasmon resonance revealed that SPC binds to both apo- and Ca2+-saturated CaM selectively, when compared with the related lysophospholipid mediators S1P, LPA (lysophosphatidic acid) and LPC (lysophosphatidylcholine). Experiments carried out with the model CaM-binding domain melittin showed that SPC dissociates the CaM-target peptide complex, suggesting an inhibitory role. The functional effect of the interaction was examined on two target enzymes, phosphodiesterase and calcineurin, and SPC inhibited the Ca2+/CaM-dependent activity of both. Thus we propose that CaM might be an intracellular receptor for SPC, and raise the possibility of a novel endogenous regulation of CaM.

A new antifungal macrolide, eushearilide, isolated from Eupenicillium shearii.

In screening for antifungal substances, a new macrolide, eushearilide (1), was isolated from Eupenicillium shearii IFM54447. The structure of 1 was established to be 24-membered macrolide having a non-conjugated diene and a choline phosphate ester moetiy on the basis of detailed investigation of NMR, UV, IR and MS spectral data. Compound 1 showed antifungal activity against various fungi and yeasts, including human pathogens Aspergillus fumigatus, Trichophyton spp. and Candida spp.

Endocannabinoid metabolism in the absence of fatty acid amide hydrolase (FAAH): discovery of phosphorylcholine derivatives of N-acyl ethanolamines.

Lipid transmitters are tightly regulated by a balance of biosynthetic and degradative enzymes. Termination of the activity of the N-acyl ethanolamine (NAE) class of lipid-signaling molecules, including the endocannabinoid anandamide (AEA), is principally mediated by the integral membrane enzyme fatty acid amide hydrolase (FAAH) in vivo. FAAH(-/-) mice are highly sensitized to the pharmacological effects of AEA; however, these animals eventually recover from AEA treatment, implying the existence of alternative routes for NAE metabolism. Here, we have pursued the characterization of these pathways by profiling the metabolome of FAAH(-/-) mice treated with AEA. Multiple AEA-induced metabolites were observed in brains from FAAH(-/-) mice, including a major product with a mass shift of +165 Da (m/z 513). The structure of this product was determined to be O-phosphorylcholine (PC)-AEA. Analysis of untreated mice identified PC-NAEs as endogenous constituents of the central nervous system (CNS) that were highly elevated in FAAH(-/-) animals. PC-NAEs were very poor substrates for FAAH; however, a vanadate-sensitive enzymatic activity was detected in brain membranes that converted PC-NAEs back to their parent NAEs. The choline-specific phosphodiesterase NPP6 was identified as a candidate enzyme responsible for this activity. These data indicate the presence of a complete metabolic pathway for the production and degradation of PC-NAEs in the CNS that constitutes an alternative route for endocannabinoid metabolism.

Separation and quantification of alkylphosphocholines by reversed phase high performance liquid chromatography.

Alkylphosphocholines represent a new class of drugs with remarkable antineoplastic and antiprotozoal activity. For instance, hexadecylphosphocholine has been approved for the topical treatment of skin metastasis. In addition, it was successfully studied in India for the treatment of leishmaniasis. Different phase-I and phase-II-trials resulted in cure rates of more than 97%. To optimize antitumor or antiprotozoal activity, we have prepared alkylphosphocholines differing in chain length and unsaturation. For the qualitative and quantitative analysis of these longer chain analogues, we have used isocratic high performance liquid chromatography. The separation of the alkylphosphocholines with different chain lengths in this reversed phase HPLC system was achieved on a YMC-TMS column with a mobile phase consisting of methanol-water (85:15; v/v) at a flow rate of 1.0 ml/min. Furthermore the cis-/trans-isomers such as oleylphosphocholine and elaidylphosphocholine were clearly separated on a YMC-C8 column with a methanol-water mixture (80:20; v/v) as mobile phase. In the described reversed phase HPLC systems simple refractive index detection and UV detection allow the sensitive and quantitative determination of alkylphosphocholines. These methods are very important for reproducible identification and quantitative determination of saturated and mono-unsaturated alkylphosphocholines with alkyl residues containing up to 25 carbon atoms.

Synthetic analogues of irlbacholine: a novel antifungal plant metabolite isolated from Irlbachia alata.

Irlbacholine and a series of related analogues were synthesized and their antifungal activities against Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus were assessed. The natural bisphosphocholine, irlbacholine, was the most potent compound, its 22-carbon chain length appearing to be optimal.

Preparation of a naturally occurring D-erythro-(2S, 3R)-sphingosylphosphocholine using Shewanella alga NS-589.

Sphingosylphosphocholine, an N-deacylated derivative of sphingomyelin, has been found to be involved in many cellular events. This paper describes a new method for preparation of a D-erythro-sphingosylphosphocholine, which is naturally occurring but difficult to prepare by chemical methods, using marine bacteria as a biocatalyst. When cultured with Shewanella alga NS-589 in synthetic medium, sphingomyelin was found to be efficiently converted by sphingomyelin deacylase to sphingosylphosphocholine. Sphingosylphosphocholine was purified with a high yield from the culture supernatant and identified to be a D-erythro-(2S,3R)-isomer containing d18:1 sphingenine as a long-chain base by fast atom bombardment mass spectrometry and NMR analyses.

Structure of a novel phosphocholine-containing aminoglycoglycerolipid of Mycoplasma fermentans.

Mycoplasma fermentans is thought to be a pathogen of rheumatoid arthritis or a cofactor of AIDS (acquired immunodeficiency syndrome). To elucidate the possible involvement of membrane constituents in the pathogenesis of these diseases, we studied its lipid components. Several alkali labile glycophospholipids were detected and named glycoglycerophospholipids (GGPLs). Previously, we purified and determined the structure of one of them as 6'-O-phosphocholine-alpha-glucopyranosyl-(1'-3)-1,2-diacyl-sn-glycerol (GGPL-I). The present paper describes the purification and structural characterization of GGPL-III, the major GGPL of M. fermentans using 1H-, 13C- and 31P-nuclear magnetic resonance spectroscopy, and mass-spectroscopy as 1"-phosphocholine,2"-amino dihydroxypropane-3"-phospho-6'-alpha-glucopyranosyl-(1'-3)-1,2-dia cyl-glycerol.

[Proton magnetic resonance spectroscopy of excised pig liver].

After evaluating ex vivo pig liver by 1H magnetic resonance spectroscopy (1H-MRS) using a 1.5 Tesla super-conducting unit, the assignment of peaks was reevaluated in vitro using a 9.4 Tesla superconducting unit. The portal vein was cannulated and perfused by cooled Euro-Collins solution, and pig liver was removed and preserved in the solution. Five to 8 g of the liver was excised before and after preservation, then extracted by perchloric acid (PCA). In 1H-MRS of PCA extracted pig liver, large peaks of fatty acid disappeared, while peaks of the choline group from 3.2-3.3 ppm were clearly observed. Using high performance liquid chromatography (HPLC) and 31P (phosphorus)-MRS, the 3.23 ppm peak was determined as glycerophosphorylcholine (GPC), which diminished after preservation. The chronological change of GPC in PCA extracted pig liver was able to be observed by 1H-MRS. GPC peak may play a role as an indicator in evaluating preserved liver by 1H-MRS.

Isolation and identification of a choline-linked mannobiose in the glycoproteins of Fusarium sp. M7-1.

An unidentified oligosaccharide was isolated from an oligomer mixture derived by alkaline borohydride treatment from glycoproteins of Fusarium sp. M7-1. The isolated compound was identified as O-alpha-D-Mannopyranosyl (1-->2)-D-Mannitol-6-phosphocholine by NMR and Ms spectrometry.

Phosphocholine-bonded galactosylceramides having a tri-unsaturated long-chain base from the clam worm, Marphysa sanguinea.

Seven glycosphingolipids were obtained in the pure state from the clam worm, Marphysa sanguinea, by preparative HPLC with a reversed-phase column in a recycling mode. Their structures were elucidated based on detailed analyses of 1H- and 13C-NMR spectra of the intact compounds. The position and the geometry of double bonds in the long-chain base (LCB) were determined by the two-dimensional NMR (COSY, NOESY, and HMBC) analyses. All compounds are phosphocholine-bonded monogalactosylceramides and two of them have tri-unsaturated long-chain bases.

Identification of a naturally occurring methyl-ester of phosphate, methyl-phosphorylcholine (methyl-2-(N,N,N trimethylamino) ethyl phosphate), in the eggs of the sea urchin S. purpuratus.

A novel metabolite of choline, phosphorylcholine methyl ester, has been identified in the eggs of S. purpuratus wherein it is present at approximately 1 mM concentration. To the best of our knowledge, this is the first instance of a phosphoryl-methyl-ester to be observed in nature. The compound appears to be species specific, since it has not been observed in other species such as L. pictus and P. depressus. In S. purpuratus its distribution is confined to the ovary, eggs and embryos, and is absent from young animals following metamorphosis.

Analysis of the water-soluble products of phosphatidylcholine breakdown by ion-exchange chromatography. Bombesin and TPA (12-O-tetradecanoylphorbol 13-acetate) stimulate choline generation in Swiss 3T3 cells by a common mechanism.

A method for the rapid and quantitative separation of glycerophosphocholine, choline phosphate and choline upon ion-exchange columns is described. The method has been utilized to examine the stimulation of phosphatidylcholine breakdown in quiescent Swiss 3T3 cells in response to bombesin and 12-O-tetradecanoylphorbol 13-acetate (TPA). The stimulated generation of choline is shown to precede that of choline phosphate, with no effect upon glycerophosphocholine levels; but was attenuated in cells in which protein kinase C activity was down-regulated. The results thus suggest that stimulation of the cells with either bombesin or TPA activates phospholipase D-catalysed phosphatidylcholine breakdown by a common mechanism involving the activation of protein kinase C.

Partial synthesis and properties of a series of N-acyl sphingomyelins.

A series of sphingomyelins (SM) with different chain length fatty acids (C14:0, C16:0, C18:0, C20:0, C22:0, and C24:0) N-linked to the primary amino group of sphingosine have been synthesized starting with bovine brain SM. Two different acid hydrolysis procedures, butanolic HCl (H. Kaller, 1961. Biochem. Z. 334: 451-456) and methanolic HCl (R.C. Gaver and C.C. Sweeley. 1965. J. Am. Oil Chem. Soc. 42: 294-298), were used and the resultant sphingosylphosphocholine (SPC) was converted to SM using two acylation methods: using fatty acid imidazolide to yield the O-acyl, N-acyl SPC, followed by mild alkaline hydrolysis for selective deacylation at the O-acyl linkage, and selective acylation at the amino group of SPC using the free fatty acid in the presence of dicyclohexylcarbodimide. Following chromatographic purification, N-acyl SM were obtained in high yield (80-90%), and were characterized by a combination of thin-layer chromatography, high performance liquid chromatography, chemical analysis, optical rotation, circular dichroism, infrared spectroscopy, 13C NMR, and sphingosine base analysis. The N-acyl SM were chemically homogeneous with respect to fatty acid composition and the sphingosine base composition resembled that of the starting bovine brain SM. However, as a consequence of the epimerization at C-3 of SPC in both acid hydrolysis procedures, the resulting N-acyl SM consisted of mixtures of D-erythro and L-threo sphingomyelins. By differential scanning calorimetry hydrated C14:0 to C24:0 SM exhibited gel-liquid crystal transitions in the range 30-50 degrees C but the chain length dependence was complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of complement in C-reactive protein positive sera by phosphorylcholine-bearing component isolated from parasite extract.

Phosphorylcholine-bearing component levels in extracts of various parasites were determined by a capillary precipitin test using anti-phosphorylcholine Ig A myeloma protein. TEPC-15. Phosphorylcholine was demonstrated as a structural component not only in nematodes but also in trematodes and cestodes. The phosphorylcholine-bearing component was isolated from an extract of Toxocara canis larvae using a TEPC-15-Sepharose 4B column. The component reacted with C-reactive protein in sera to form one precipitin line in immunoelectrophoresis. The component provided two Brilliant Coomassie Blue positive bands in SDS-polyacrylamide gel electrophoresis. It reacted with C-reactive protein to activate complement in serum.