Identification of very long chain unsaturated fatty acids from Ximenia oil by atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy

A method is described for the enrichment of very long chain unsaturated fatty acids from total fatty acids of Ximenia oil and their identification as picolinyl esters by means of liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The method is based on the use of preparative reversed phase HPLC and their subsequent identification by microbore LC-MS/APCI. The combination of these two techniques was used to identify unusual unsaturated VLCFAs up to tetracontenoic acid. All four positional isomers of tetratriacontenoic acid were also synthesized to unambiguously confirm their structure.     

Chromatographic determination of optical configuration of 3-hydroxy fatty acids composing microbial surfactants

Abstract The chromatographic determination of the optical configuration of 3-hydroxy fatty acids of microbial surfactants was achieved in chiral high pressure liquid chromatography (HPLC) by injecting 3,5-dinitroaniline-derivatives of crude hydrolysates (less than 1 mg). Serrawettin W2, a surface-active cyclodepsipeptide of Serratia marcescens , was shown to contain d -3-hydroxydecanoic acid. Rubiwettin R1 and RG1, surface active glycolipid and linked fatty acids of Serratia rubidaea , were shown to contain d -3-hydroxytetradecanoic acid and d -3-hydroxydecanoic acid. The new method does not require purified sample or authentic optical isomers, and could be useful in the structural analysis of microbial lipids.     

Optical Configuration Analysis of Hydroxy Fatty Acids in Bacterial Lipids by Chiral Column High-Performance Liquid Chromatography

Through the adoption of a chiral stationary phase in high-performance liquid chromatography and a simple derivatization method for hydroxy fatty acids, it became easy to separate and identify the optical isomers of 2- and 3-hydroxy fatty acids composing several kinds of microbial lipids. The 2- and 3-hydroxy fatty acids were converted with dinitrophenyl isocyanate to their 3, 5-dinitrophenyl urethane derivatives (DU-derivatives), which were analyzable by HPLC using a chiral column. By varying the composition of an eluent, separation of the DU-derivatives of hydroxy fatty acids differing in optical configuration, chain length and position of hydroxyl group was achieved. The general elution orders of these DU-derivatives were determined with authentic 2- and 3-hydroxy fatty acids. Small amounts (~300 μg) of ornithine-containing lipids isolated from the Serratia marcescens strains were examined by this method to identify 3-hydroxy fatty acids of the lipids as D isomers.     

Diacylglycerols interfere in normal phase HPLC analysis of lipoxygenase products of docosahexaenoic or arachidonic acids

A methodological problem with the normal phase high performance liquid chromatography (HPLC) of hydroxylated products of docosahexaenoic and arachidonic acids is described. Diacylglycerols present in lipid extracts of rat retina co-elute with monohydroxy derivatives of docosahexaenoic or arachidonic acid, when samples are applied to uPorosil columns and eluted with hexane/isopropanol/acetic acid. Analysis of fatty acid composition of diacylglycerols which were acetone-extracted from the incubation medium showed a profile similar to diacylglycerols extracted from the tissue by hexane/isopropanol, although acetone extraction resulted in extremely variable recovery of diacylglycerols. This co-elution of diacylglycerols with monohydroxy polyunsaturated fatty acids can lead to a significant error in estimation of lipoxygenation activity by conversion of radiolabeled precursors, because the incorporation of fatty acids into diacylglycerols is very active in many tissues. An alternative extraction method and reverse phase HPLC procedures that result in the complete separation of hydroxy fatty acids and diacylglycerols are described.     

Resolution of enantiomers of hydroxyeicosatetraenoate derivatives by chiral phase high-pressure liquid chromatography

A new method was developed for analyzing the steric configuration of hydroxyeicosatetraenoates (HETEs) and other hydroxy fatty acids. Racemic HETE methyl esters were reacted with either benzoyl or naphthoyl chloride in pyridine and the resulting aromatic ester derivatives purified by reversed phase HPLC and subsequently chromatographed on a chiral stationary phase HPLC column [(R)-(-)-N-3,5-dinitrobenzoyl-alpha-phenylglycine)]. In contrast to the enantiomers of the underivatized HETE methyl esters which were only partially resolved, the enantiomers of their aromatic ester derivatives were completely separable on this chiral phase. Chiral HETEs can be retrieved from the aromatic derivatives by alkaline hydrolysis. Thus, this method has both analytical and preparative applications.     

Determination of stereochemistry in the fatty acid hydroperoxide products of lipoxygenase catalysis

High-performance liquid chromatography has been found to be an effective method for the determination of absolute configuration in the products of the lipoxygenase-catalyzed oxygenation of polyunsaturated fatty acids. Methyl esters of fatty acid hydroperoxides that had been reduced to the corresponding alcohols were converted into (+)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid esters. Enantiomeric alcohols were converted into diastereomeric esters that were readily resolved by normal-phase HPLC. The instrumental requirements for the technique are an isocratic HPLC and a uv absorbance monitor. The method was found to be effective in the determination of stereochemistry in the products derived from the action of plant lipoxygenases on linoleic acid. In addition, the chromatography of the derivatives obtained from lipoxygenase catalysis on arachidonic acid was found to be effective for the assignment of stereochemistry in those products. A comparison of the chromatography of these derivatives with that for the corresponding menthyloxycarbonyl derivatives demonstrated the superiority of this approach for the resolution of the diastereomeric pairs. The technique was applied to the determination of stereochemistry in the products derived from soybean lipoxygenase isoenzymes under a variety of experimental conditions.     

Synthesis and applications of stereospecifically (3)H-labeled arachidonic acids as mechanistic probes for lipoxygenase and cyclooxygenase catalysis

Stereospecifically (3)H-labeled substrates are useful tools in studying the mechanism of hydrogen abstractions involved in the oxygenation of polyunsaturated fatty acids. Here, we describe modified methods for the synthesis of arachidonic acids labeled with a single chiral tritium on the methylene groups at carbons 10 or 13. The appropriate starting material is a ketooctadecanoic acid which is prepared from an unsaturated C18 fatty acid precursor or by total synthesis. The (3)H label is introduced by NaB(3)H(4) reduction and the resulting tritiated hydroxy fatty acid then is tosylated, separated into the enantiomers by chiral phase HPLC, and subsequently transformed into stearic acids. A variety of stereospecifically labeled unsaturated fatty acids are obtained using literature methods of microbial transformation with the fungus Saprolegnia parasitica. Two applications are described: (i) In incubations of [10S-(3)H]- and [10R-(3)H]arachidonic acids in human psoriatic scales we show that a 12R-lipoxygenase accounts not only for synthesis of the major product 12R-HETE, but it contributes also, through subsequent isomerization, to the minor amounts of 12S-HETE. (ii) The [10R-(3)H]- and [10S-(3)H]arachidonic acids were also used to demonstrate that prostaglandin ring formation by cyclooxygenases does not involve carbocation formation at C-10 of arachidonic acid as was hypothesized recently.     

Stereochemical analysis of hydroxylated docosahexaenoates produced by human platelets and rat brain homogenate

The stereochemical configuration of hydroxylated products of docosahexaenoic acid (22:6w3) formed by human platelets and rat brain homogenate were characterized for the first time. Chiral phase HPLC was employed along with autooxidized 22:6w3 as reference material. The 14- and 11-hydroxy 22:6w3 (HDHE) products produced by human platelets were in the S configuration. Rat brain homogenate produced all of the ten possible positional isomers when incubated with 22:6w3. Their retention behavior on the reversed and chiral phase HPLC columns and GC/MS/EI analysis indicated that they were 20-, 17-, 16-, 14-, 13-, 11-, 10-, 8-, 7- and 4-HDHE. However, stereochemical analysis revealed that each positional isomer was a racemic mixture, suggesting that these were not formed by lipoxygenation but mainly by peroxidation process.     

Synthesis of four isomers of parinaric acid

A simple and reliable method for synthesizing four isomers of parinaric acid from alpha-linolenic acid (ALA) in high yields is described. The methylene-interrupted, cis triene system (1,4,7-octatriene) of ALA and common to other naturally occurring polyunsaturated fatty acids was transformed to a conjugated tetraene system (1,3,5,7-octatetraene). The synthesis involves bromination of ALA using 0.l M Br(2) in a saturated solution of NaBr in methanol, esterification of the fatty acid dibromides, double dehydrobromination by 1,8-diazabicyclo[5.4.0]undec-7-ene and saponification of the conjugated esters to a mixture of free conjugated acids. Addition of one molecule of bromine to the 12,13-double bond of ALA and subsequent dehydrobromination produces alpha-parinaric acid (9Z,11E,13E,15Z-octadecatetraenoic acid); addition of Br(2) to the 9,10-double bond or 15,16-double bond and then dehydrobromination and rearrangement yields 9E,11E,13E,15Z-octadecatetraenoic or 9E,11E,13E,15Z-octadecatetraenoic acids, respectively. The mixture of parinaric acid isomers is obtained in 65% yield, and the isomers can be purified by preparative HPLC; alternatively, the isomers can be converted by base catalyzed cis-trans isomerization (or by treatment with I(2)) to exclusively beta-parinaric acid (9E,11E,13E,15E-octadecatetraenoic acid). The various parinaric acid isomers were characterized by (1)H NMR, (13)C NMR, UV, GLC, HPLC and mass spectrometry.     

Determination of optical purity of 3,5-dimethoxybenzoyl-leucine diethylamide by chiral chromatography and 1H and 13C NMR spectroscopy

(R)-N-3,5-dinitrobenzoyl (DNB) leucine derived chiral selector was used as an HPLC chiral stationary phase for the resolution of various racemic amino acids derivatives. In this study, determination of optical purity of an amino acid derivative was performed by chiral high performance liquid chromatography and 1H and 13C NMR spectroscopy by using the DNB leucine derived chiral selector. The accuracy and precision of each optical purity value are calculated and the data are compared to each other.     

A NMR shift method for determination of the enantiomeric composition of hydroperoxides formed by lipoxygenase.

A method is presented for determination of the enantiomeric composition of hydroxyperoxides formed by enzymic oxygenation of unsaturated fatty acids. After reduction of the hydroperoxy group with NaBH4, and esterification, the positional isomers of the resulting hydroxy compounds are separated by high performance liquid chromatography. The latter are subsequently subjected to a chiral derivatization to form diastereomeric alpha-methoxy-alpha-trifluoromethylphenylacetate esters. Determination of the diastereomeric composition by a NMR shift experiment furnishes the enantiomeric composition of the parent hydroperoxides. The method has been applied to the hydroperoxides formed by incubation of linoleic acid by corn germ or soybean lipoxygenase. Our results indicate that under the conditions used the hydroperoxides are mainly enantiospecifically formed.     

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid.

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.     

High Performance Liquid Chromatography of Hydroperoxides Formed by Autoxidation of Vegetable Oils

Trilinoleoylglycerol (TL) was autoxidized at 37°C in the dark. Monohydroperoxides (MHP) obtained from the oxidized products were analyzed by high performance liquid chromatography (HPLC). Several peaks which appeared in the chromatogram were identified by infrared (IR), gas chromatography mass spectrometry (GC-MS) and enzymatic hydrolysis. Some positional and geometrical isomers of their hydroperoxy fatty acid components were separated using both absorption and reversed phase systems. Furthermore, 1-hydroperoxylinoleoyl-2,3-dilinoleoyl-glycerol and 1,3-dilinoleoyl-2-hydroperoxylinoleoylglycerol were partly separated by HPLC using an absorption system. MHP obtained from autoxidized corn oil, safflower oil and soybean oil were separated into some peaks by HPLC, although resolution into the individual isomers was incomplete. When oxidized oils were subjected to HPLC analysis directly, a linear relationship was observed between the peak areas of MHP and peroxide value in the range of 10 ~ 50 meq/kg.     

Quantitative effects of unsaturated fatty acids in microbial mutants. VII. Influence of the acetylenic bond location on the effectiveness of acyl chains.

The ability of a series of 18 carbon acetylenic fatty acids to fulfill the unsaturated fatty acid requirements of Escherichia coli and Saccharomyces cerevisiae was investigated. Despite their high melting points (greater than 40 degrees C), several isomers of the acetylenic fatty acids were as efficient or more efficient in supporting growth than the analogous fatty acid having a cis-double bond. The efficiencies of the different positional isomers in supporting cell proliferation varied from essentially 0 cells per fmol for the 2-5 and 13-17 isomers to high values when the acetylenic bond was near the center of the chain: e.g. 45 E. coli and 5.5 S. cerevisiae cells/fmol for the 10 isomer. A striking ineffectiveness of the 9 isomer was observed with E. coli. The 7, 8 and 10 isomers were at least 10-fold more efficient than any of the other positional isomers in supporting the growth of E. coli. In contrast, the 9 isomer was among the most effective acetylenic fatty acids tested with the yeast mutant. Chromatographic analysis of the extracted lipids indicated that each of the acetylenic isomers tested (except delta2 and delta3) could be esterified by the prokaryotic and eukaryotic microorganisms. The content of unsaturated plus cyclopropane acids observed when growth ceased in E. coli cultures supplemented with growth-limiting concentrations of the acetylenic fatty acids ranged from approx. 15 mol% for the 8 isomer to approx. 35 mol% for the 14 and 17 isomers. The 8-11 isomers were observed to be esterified predominantly at the two position in phosphatidylethanolamine of E. coli and in phosphatidylcholine of S. cerevisiae.     

High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry

Historically considered to be simple membrane components serving as structural elements and energy storing entities, fatty acids are now increasingly recognized as potent signaling molecules involved in many metabolic processes. Quantitative determination of fatty acids and exploration of fatty acid profiles have become common place in lipid analysis. We present here a reliable and sensitive method for comprehensive analysis of free fatty acids and fatty acid composition of complex lipids in biological material. The separation and quantitation of fatty acids are achieved by capillary gas chromatography. The analytical method uses pentafluorobenzyl bromide derivatization and negative chemical ionization gas chromatography-mass spectrometry. The chromatographic procedure provides base line separation between saturated and unsaturated fatty acids of different chain lengths as well as between most positional isomers. Fatty acids are extracted in the presence of isotope-labeled internal standards for high quantitation accuracy. Mass spectrometer conditions are optimized for broad detection capacity and sensitivity capable of measuring trace amounts of fatty acids in complex biological samples. .     

Recent advances in methods of direct optical resolution

Very great advances have been made in the field of direct optical resolution of organic compounds by chromatographic techniques. Chiral capillary gas chromatography now permits a determination of the enantiomeric composition of a few nanograms of a compound present in a mixture of many others. Coupled with high resolution mass spectrometry the technique will additionally permit structural elucidation; of great interest in pheromone research and related areas. Analytical separations of enantiomers are now also carried out by high-performance liquid chromatography (HPLC) methods based on a variety of principles. Basically, two main types are used, differing as to whether the mobile phase has to be a chiral medium or not. Two-dimensional HPLC, whereby compounds separated on a non-chiral column are progressively and automatically transferred to a chiral column for optical resolution, has been used successsfully for chiral amino acid separations. Many different chiral sorbents for preparative LC and HPLC resolutions have been prepared; some of these are now used in columns capable of producing pure enantiomers from a given racemate at a rate of the order of one gram/hour in continuous, automatic HPLC procedures. Apart from all important applications of these results of optical resolution technology, an increased knowledge of the underlying chiral recognition phenomena responsible for enantioselection has also been achieved.     

Soybean lipoxygenase-1 oxygenates synthetic polyenoic fatty acids with an altered positional specificity: evidence for inverse substrate alignment

The positional specificity is the decisive enzyme property for classification of lipoxygenases and for the currently used lipoxygenase nomenclature. It has been reported before that soybean lipoxygenase-1, which oxygenates polyenoic fatty acids at alkaline pH to the corresponding n - 6 hydroperoxy derivative, exhibits a different positional specificity when either the reaction conditions or the substrate structure is altered. To investigate the impact of structural substrate modifications on the positional specificity of this enzyme and to force an inverse substrate binding, we synthesized arachidonic acid analogues modified at the omega-terminus. Care was taken that the double bond system remained unchanged so that hydrogen abstraction from all three bisallylic methylenes was theoretically possible. We found that omega-modification of arachidonic acid leads to an impaired substrate affinity and a reduced reaction rate, but we did not detect any 5-lipoxygenation products, suggesting that structural modification of the omega-end may not be sufficient to force an inverse substrate orientation. However, when both ends of the fatty acid chain (omega-terminus and free carboxylate) were modified simultaneously, a considerable share of 5-lipoxygenation products was detected. These results indicate that introduction of polar or bulky groups at the methyl terminus of polyenoic fatty acids was not sufficient to force an inverse substrate orientation. However, simultaneous introduction of an omega-OH group and methylation of the carboxylate led to formation of significant 5-lipoxygenation products, suggesting an inverse head to tail substrate orientation.     

Formation of monohydroxy derivatives of arachidonic acid, linoleic acid, and oleic acid during oxidation of low density lipoprotein by copper ions and endothelial cells.

An important event in the formation of atherosclerotic lesions is the uptake of modified low density lipoprotein (LDL) by macrophages via scavenger receptors. Modification of LDL, which results in its recognition by these receptors, can be initiated by peroxidation of LDL lipids. The first step in this process is the formation of monohydroperoxy derivatives of fatty acids, which are subsequently degraded to the corresponding monohydroxy compounds, or to a variety of secondary oxidation products. In order to understand this process more completely, we have developed a mass spectrometric procedure to measure the amounts of specific hydroperoxy/hydroxy fatty acids formed by oxidation of the major unsaturated fatty acids in human LDL, oleic acid, linoleic acid, and arachidonic acid. Oxidation of human LDL in the presence of a relatively strong stimulus (20 microM CuSO4) resulted in very large increases in the amounts of the major monohydroxy derivatives of linoleic acid (9- and 13-hydroxy derivatives) and arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxy derivatives) in LDL lipids in the early stages of the reaction. After 20 h, the amounts of these products declined due to substrate depletion, but large amounts of monohydroxy derivatives of oleic acid (8-, 10-, and 11-hydroxy derivatives) were detected. Although thiobarbituric acid-reactive substances clearly increased under these conditions, the changes were not nearly so dramatic as those observed for monohydroxy fatty acids. Oxidation of LDL in the presence of endothelial cells, a much milder stimulus, resulted in 2.5- to 3-fold increases in the amounts of monohydroxy derivatives of linoleic and arachidonic acids, as well as thiobarbituric acid-reactive substances, with more modest increases in the amounts of hydroxylated derivatives of oleic acid. There was little positional specificity in the oxidation of the above fatty acids in the presence of either stimulus, suggesting that the formation of these products proceeds primarily by lipid peroxidation, rather than by catalysis by lipoxygenases. However, an important role for lipoxygenases in the initiation of these reactions cannot be excluded. In conclusion, oxidation of LDL in the presence of copper ions or endothelial cells results in the formation of a large number of monohydroxy derivatives of oleic, linoleic, and arachidonic acids. The relative amounts of products formed from each of these fatty acids depends on the strength of the stimulus as well as the incubation time.     

Hydroperoxy-arachidonic acid mediated n-hexanal and (Z)-3- and (E)-2-nonenal formation in Laminaria angustata

In higher plants, C6 and C9 aldehydes are formed from C18 fatty acids, such as linoleic or linolenic acid, through formation of 13- and 9-hydroperoxides, followed by their stereospecific cleavage by fatty acid hydroperoxide lyases (HPL). Some marine algae can also form C6 and C9 aldehydes, but their precise biosynthetic pathway has not been elucidated fully. In this study, we show that Laminaria angustata, a brown alga, formed C6 and C9 aldehydes enzymatically. The alga forms C9 aldehydes exclusively from the C20 fatty acid, arachidonic acid, while C6 aldehydes are derived either from C18 or from C20 fatty acid. The intermediates in the biosynthetic pathway were trapped by using a glutathione/glutathione peroxidase system, and subjected to structural analyses. Formation of (S)-12-, and (S)-15-hydroperoxy arachidonic acids [12(S)HPETE and 15(S)HPETE] from arachidonic acid was confirmed by chiral HPLC analyses. These account respectively for C9 aldehyde and C6 aldehyde formation, respectively. The HPL that catalyzes formation of C9 aldehydes from 12(S)HPETE seems highly specific for hydroperoxides of C20 fatty acids.