Covalent interaction of chloroacetic and acetic acids with cholesterol

The covalent interaction of chloroacetic acid with rat liver lipids was studied in vivo. Rats were given a single oral dose (8.75 mg/kg, 50 microCi) of 1-[14C]chloroacetic acid and sacrificed after 24 hours. Lipids extracted from the livers were separated into neutral lipids and phospholipids by solid-phase extraction using sep-pak silica cartridges. The neutral lipid fraction was further fractionated by preparative thin-layer chromatography followed by reverse-phase high-performance liquid chromatography. The fraction corresponding to the retention time of standard cholesteryl chloroacetate gave a pseudomolecular ion peak at m/z 480/482 ratio: (3:1) on ammonia chemical ionization mass spectrometry, and the fragmentation pattern was found to be similar to that of the standard sample. Under similar conditions, acetic acid resulted in the formation of cholesteryl acetate. The effect of such conjugation reactions on the cell membrane and their contribution to toxicity is presently unknown.     

N-acetyl-9-O-L-lactylneuraminic acid, a new acylneuraminic acid from bovine submandibular gland.

The acylneuraminic acid fraction, obtained on mild acid hydrolysis of glycoproteins from bovine submandibular glands, contains approximately 2% N-acety-9-O-L-lactylneuraminic acid. The compound has been isolated and purified by ion-exchange and cellulose column chromatography. The structure has been elucidated using thin-layer chromatography, colorimetry, gas-liquid chromatography/mass spectrometry, periodate oxidation and specific lactate dehydrogenases. An evaluation of the different analytical methods is given.     

Isolation of 9-hydroxy-delta-tetradecalactone from lipid A of Pseudomonas diminuta and Pseudomonas vesicularis

A lipid component was isolated from the fatty acid fraction of acid hydrolysates of lipid A derived from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 lipopolysaccharides. By structural analysis of the lipid and its trimethylsilyl and acetyl derivatives by thin-layer chromatography, gas chromatography-mass spectrometry, mass spectrometry, infrared spectrometry and 13C-NMR, it was identified as 9-hydroxy-delta-tetradecalactone.     

Chemical characterization of recombinant human leukocyte interferon A using fast atom bombardment mass spectrometry

Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.     

Isolation of 9-hydroxy-δ-tetradecalactone from lipid A of Pseudomonas diminuta and Pseudomonas vesicularis

Abstract A lipid component was isolated from the fatty acid fraction of acid hydrolysates of lipid A derived from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 lipopolysaccharide. By structural analysis of the lipid and its trimethylsilyl and acetyl derivatives by thin-layer chromatography, gas chromatography-mass spectrometry, mass spectrometry, infrared spectrometry and ¹³C-NMR, it was identified as 9-hydroxy-δ-tetradecalactone.     

Isolation and identification of ferrioxamine G and E inHafnia alvei

Summary Under conditions of iron-deprivationHafnia alvei (Enterobacteriaceae) produces ferrioxamine G as the principal siderophore. Maximum hydroxamate siderophore production occurred at medium iron limitation. The ferrioxamines were extracted, purified by gel filtration and chromatography on silica gel yielding a major and a minor siderophore fraction. The minor siderophore fraction contained three siderophores, among which ferrioxamine E could be identified by HPLC and FAB mass spectrometry. Reductive hydrolysis of the ferrioxamine G fraction yielded succinic acid and a mixture of diaminopentane and diaminobutane, as determined by gas-liquid chromatography and GLC/MS. HPLC and FAB mass spectrometry confirmed that the ferrioxamine G fraction consisted of two different species, G₁ and G₂, possessing molecular masses of 671 Da and 658 Da respectively.     

Bile acids. LXXVII. Large-scale preparation of 5 alpha-anhydrocyprinol from carp bile

An improved method of preparation of 5 alpha-anhydrocyprinol from large quantities of carp bile is reported. The following materials were obtained by this method: 5 alpha-anhydrocyprinol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol, 5 alpha-cholestane 3 alpha,7 alpha,12 alpha,26-tetrol, an unidentified sterol from the neutral lipid fraction, allocholic acid and allochenodeoxycholic acid from the bile acid fraction. Yields of 5 alpha-anhydrocyprinol and bile acids vary in different batches. The identity and purity of these materials were verified by thin-layer chromatography, gas liquid chromatography and gas chromatography-mass spectrometry.     

Streptomyces species with madurose (3-O-methyl-D-galactose) as a whole-cell sugar

Madurose, an actinomycete whole-cell sugar, was found in the strains of the genus Streptomyces: three strains of S. platensis, one strain each of S. platensis subsp. malvinus, and S. albus subsp. albus. The sugar was isolated from the hydrolysate of S. platensis IFO 14008 cells, and was identified as madurose (3-O-methyl-D-galactose) by chromatographic analyses, ¹H-NMR spectrometry, mass spectrometry as its alditol acetate, and demethylation with boron trichloride. Cell walls of the strain contained peptidoglycan and teichoic acids. ll-Diaminopimelic acid, glycine, glutamic acid, and alanine were present in the peptidoglycan fraction in molar ratios of 1.0:1.3:1.2:2.3. Madurose was detected in the teichoic acid fraction, which was composed of phosphorus, glycerol, galactose, and madurose in molar ratios of 9.3:8.5:2.9:1.0. Thus, madurose was found in the glycerol teichoic acid moiety of the cell walls of this strain.Abbreviations PC paper chromatography - TLC thin-layer chromatography - HPLC high performance liquid chromatography - GLC gas-liquid chromatography - TCA trichloroacetic acid     

New polar acid metabolites in human urine

A sequence of chromatographic methods (thin-layer chromatography, high-performance liquid chromatography and glass capillary gas chromatography) was used to separate the acid fraction of human urine. The power of this method to separate and detect previously unknown compounds and the elucidation of their final structure with mass spectrometry is exemplified by the identification of N-acetyl-2-aminooctanoic acid as a metabolic compound in the urine of healthy individuals.In addition, the conjugate of glycine with indolepropionic acid, N-formylanthranilic acid, succinoylphenylalanine, δ-hydroxyvaleric acid, δ-hydroxycapric acid, 3-hydroxyadipic acid, and higher homologues were detected in a polar fraction of human urine.     

The determination of phytosphingosine-containing globotriaosylceramide from human kidney in the presence of lactosylceramide

Globotriaosylceramide, the natural substrate of alpha-galactosidase A (the enzyme deficient in Fabry's disease) was prepared from human kidney by repeated medium pressure chromatography on Lichroprep Si 60 (E. Merck) before and after peracetylation. The apparently homogeneous preparation migrating as a single band on HPTLC was analysed by fast atom bombardment mass spectrometry and 1H-NMR at 500 MHz. It was found that in this fraction two major molecular species were comigrating: Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide with nervonic and lignoceric acid linked to phytosphingosine and Gal beta 1-4Glc beta 1-1 ceramide with palmitic acid linked to sphingosine.     

Structural analysis of neutral glycosphingolipids from Ascaris suum adults (Nematoda: Ascaridida)

The neutral glycosphingolipid fraction from adults of the pig parasitic nematode, Ascaris suum, was resolved into four components on thin-layer chromatography. The high-performance liquid chromatography-isolated components were structurally analysed by: methylation analysis; exoglycosidase cleavage; gas-liquid chromatography/mass spectrometry; liquid secondary-ion mass spectrometry; and, in particular, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their chemical structures were determined as: Glc(β1-1)ceramide, Man(β1-4)Glc(β1-1)ceramide, GlcNAc(β1-3)Man(β1-4)Glc(β1-1)ceramide and Gal(α1-3)GalNAc(β1-4)GlcNAc(β1-3)Man(β1-4)Glc(β1-1)ceramide; and were characterized as belonging to the arthro-series of protostomial glycosphingolipids. No glycosphingolipid component corresponding to ceramide tetrasaccharide was detected during these analyses. The ceramide composition of the parent glycosphingolipids was dominated by the 2-(R)-hydroxy C24:0 fatty acid, cerebronic acid, and C17 sphingoid-bases: 15-methylhexadecasphing-4-enine and 15-methylhexadecaphinganine in approximately equal proportions. The component ceramide monohexoside was characterized by an additional 15-methylhexadecaphytosphingosine. Abbreviations: CDH, ceramide dihexoside; Cer, ceramide; CMH, ceramide monohexoside; CPH, ceramide pentahexoside; CTH, ceramide trihexoside; CTetH, ceramide tetrahexoside; Hex, hexose; HexNAc, N-acetylhexosamine; HPTLC, high-performance thin-layer chromatography; LSIMS, liquid secondary-ion mass spectrometry; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; N-, Nz- and A-glyco(sphingo)lipids, neutral, neutralzwitterionic and acidic glyco(sphingo)lipids, respectively This revised version was published online in November 2006 with corrections to the Cover Date.     

Phospholipids of Clostridium perfringens: a reexamination

We have identified phosphatidylethanolamine as one of the major phospholipids of Clostridium perfringens by two dimensional thin layer chromatography of the intact lipids and of their deacylation products and by liquid chromatography followed by mass spectrometry of the intact neutral phospholipid fraction. The principal fatty acids of phosphatidylethanolamine are myristic acid (14:0), lauric acid (12:0), and palmitic acid (16:0) and the major molecular species are 14:0,14:0 (26.3%); 12:0,14:0 (19.0%); 14:0,16:0 (22.4%) and 16:0,16:0 (17.6%). A similar distribution of molecular species was found in the other major phospholipid, O-alanyl phosphatidylglycerol.     

Bile Acids. LXXVII. Large-Scale Preparation of 5α-Anhydrocyprinol from Carp Bile

An improved method of preparation of 5α-anhydrocyprinol from large quantities of carp bile is reported. The following materials were obtained by this method: 5a-anhydrocyprinol, 5α-cholestane-3α, 7α, 12α, 25-tetrol, 5α-cholestane3α, 7α, 12α, 26-tetrol, an unidentified sterol from the neutral lipid fraction, allocholic acid and allochenodeoxycholic acid from the bile acid fraction. Yields of 5α-anhydrocyprinol and bile acids vary in different batches. The identity and purity of these materials were verified by thin-layer chromatography, gas liquid chromatography and gas chromatography-mass spectrometry.     

Aromatic Substances in Leaves of Populus tremula as Inhibitors of Mycorrhizal Fungi

The inhibitory principles of aqueous extracts of aspen leaves, acting upon mycorrhizal fungi of forest trees, were isolated by extraction with ethyl acetate and chromatography on silicic acid. Two inhibitors were identified as benzoic acid and catechol by gas chromatography and mass spectrometry. When added to a synthetic medium, these substances had a strong inhibitory effect on the growth of different Boletus-species and a weaker inhibitory effect on litter-decomposing Marasmius-species. When the fraction of the extract which was not soluble in ethyl acetate was included in the medium, the aromatic compounds still inhibited the mycorrhizal fungi while the growth of the litter-decomposers was stimulated.     

Human cecal bile acids: concentration and spectrum

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3alpha-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3alpha-hydroxy bile acid concentration (mumol bile acid/ml cecal content) was 0.4 +/- 0.2 mM (mean +/- SD); the total 3-hydroxy bile acid concentration was 0.6 +/- 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 +/- 9%, mean +/- SD); sulfated, nonamidated bile acids were 7 +/- 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 +/- 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 +/- 16%), lithocholic (26 +/- 10%), and ursodeoxycholic (6 +/- 9), as well as their primary bile acid precursors cholic (6 +/- 9%) and chenodeoxycholic acid (7 +/- 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 +/- 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.     

Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry

A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.     

Detection of lactobacillic acid in low erucic rapeseed oil—A note of caution when quantifying cyclic fatty acid monomers in vegetable oils

The purpose of this work was to identify an unknown component which has been detected during the analysis of cyclic fatty acid monomers (CFAMs) in low erucic acid rapeseed oils (LEAR). A sample of crude LEAR was transformed into fatty acid methyl esters (FAMEs) and hydrogenated using PtO₂. The hydrogenated sample was fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and the fraction containing the CFAMs transformed into picolinyl esters. Analysing these picolinyl derivatives by gas–liquid chromatography coupled to mass spectrometry (GC–MS) showed that the unknown product observed in LEAR is the 11,12-methylene-octadecanoic acid. This cyclic fatty acid was also found in crude LEAR and in the corresponding seeds but was not detected in crude soya and sunflower oils. As this acid is present in the same fraction as CFAMs, known to be formed during heat treatment, great care must therefore be taken for not including it when quantifying CFAMs. It is thus necessary to verify by mass spectrometry the structures of the CFAMs in the isolated cyclic fatty acid fraction prior to quantification.     

Formation of leukotriene B4-coenzyme A ester by rat liver microsomes

When leukotriene B4 (LTB4) was incubated with rat liver microsomal fraction in the presence of coenzyme A (CoA) and ATP, a more polar product (compound I) was detected on reverse-phase high-performance liquid chromatography (RP-HPLC). The product was identified as LTB4-CoA ester on the basis of ultraviolet spectrometry, alkaline hydrolysis followed by RP-HPLC, and fast atom bombardment mass spectrometry (FAB-MS). The activity forming LTB4-CoA ester was localized in the microsomal fraction. The reaction was proportional to the concentration of the microsomal protein with an optimal pH of 7.5-8.0 and completely dependent on CoA and ATP. Palmitic acid and myristic acid significantly inhibited the formation.     

A soluble acyl-coenzyme A oxidase from the yeast Candida utilis.

trans-2-Enoyl-CoA and two unidentified polar compounds were synthesized from the corresponding long-chain acyl-CoA by a particle-free supernatant fraction obtained from Candida utilis. The enzyme was unreactive toward free fatty acids but desaturated all long-chain acyl CoAs tested (14:0, 16:0, 18:1, 18:2). Molecular oxygen was the only required cofactor. Phenazine methosulfate and 2,6-dichloroindophenol did not replace the requirement for oxygen. The activity was inhibited specifically by NADPH and stimulated by linoleic acid or linolenic acid. The enzyme was not active in log phase cultures, but was detected only in stationary phase cells. Introduction of the trans-2-double bond was confirmed by gas-liquid chromatography, thin-layer chromatography, mass spectrometry, catalytic hydrogenation, oxidative cleavage, and chemical reactivity of the product toward nucleophilic addition.     

Metabolism of arachidonic acid in polymorphonuclear leukocytes. Structural analysis of novel hydroxylated compounds.

Arachidonic acid was incubated with rabbit peritoneal polymorphonuclear leukocytes (glycogen-induced) and compounds obtained from ether extractions were fractionated by silicic acid column chromatography. A fraction containing several unidentified metabolites of arachidonic acid was analyzed by reversed phase-high pressure liquid chromatography. The metabolites were esterified and further purified by silicic acid high pressure liquid chromatography. The structures of the pure compounds were elucidated by infrared and ultraviolet spectrometry, ozonolysis, and gas chromatography-mass spectrometry. The following novel compounds were identified: Compound 1, 5S, 12R-dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid; Compound 2, 5S, 12S-dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid; Compound 3, 5, 6-dihydroxy-7,9,11,14-eicosatetraenoic acid; Compound 4, a diastereoisomer of the latter. Evidence for the occurrence of the delta-lactone forms of the 5,12-dihydroxy acids is also presented.