Purification of fatty acid methyl esters by high-performance liquid chromatography

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane–isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms.     

In vitro effects of sterculic acid on lipid biosynthesis in Rhodococcus opacus strain PD630 and isolation of mutants defective in fatty acid desaturation

The in vivo effects of sterculic acid methyl ester on triacylglycerol fatty acid composition in the oleaginous, hydrocarbon-degrading bacterium R. opacus strain PD630 was investigated. Sterculic acid, a cyclopropene fatty acid and an inhibitor of the stearoyl-CoA desaturase system, strongly inhibited the synthesis of monoenic fatty acids, of saturated fatty acids with more than 16 carbon atoms and of odd-numbered fatty acids when added to the culture medium. In addition, chemical mutagenesis and the application of the penicillin enrichment technique provided mutants, which were more or less completely impaired in the desaturation of long-chain fatty acids and exhibited in some cases a similar fatty acid composition like the wild-type in the presence of sterculic acid methyl ester. The implications of these findings for fatty acid metabolism in R. opacus strain PD630 are discussed.     

Microwave-assisted synthesis of deuterium labeled estrogen fatty acid esters

Deuterated analogs of estrogen fatty acid esters are needed as internal standards for isotope dilution GC/MS analyses. We have developed a rapid and efficient synthesis for 2,4,16,16-D4-estrone palmitate, stearate, oleate, linoleate, and linolenate and the corresponding 2,4,16,16,17alpha-D5-estradiol fatty acid 17-mono and 3,17-diesters using analogous fatty acid chlorides or fatty acid anhydrides and 4-(dimethylamino)pyridine under microwave irradiation. Chemoselective hydrolysis of fatty acid diesters was carried out by KOH in t-BuOH.     

Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty acid anilides

The activity of the enzyme involved in catalyzing the formation of fatty acid anilides can be measured by quantitating the fatty acid anilides formed. We have shown earlier that oleic acid is the most preferred substrate among other fatty acids studied for the conjugation with aniline. The reaction product (oleyl anilide) could be separated by thin-layer chromatography (TLC) and then quantified by reversed-phase high-performance liquid chromatography (HPLC). Using [1-¹⁴C]oleic acid as substrate, the fatty acid anilide forming activity can be determined in a single step by TLC analysis. The conventional TLC methods used for the separation of the fatty acid esters, however, could not resolve oleyl anilide from the residual [1-¹⁴C]oleic acid. Therefore, a simple and reliable TLC method was developed for the separation of oleyl anilide from oleic acid using a freshly prepared solvent consisting of petroleum ether–ethyl acetate–ammonium hydroxide (80:20:1, v/v). Using this solvent system the relative flow (R_f) values were found to be 0.54 for oleyl anilide and 0.34 for aniline, whereas oleic acid remained at the origin. The TLC procedure developed in the present study could be used to determine the fatty acid anilide forming activity using [1-¹⁴C]oleic or other fatty acids as substrate and was also found suitable for the analysis of fatty acid anilides from the biological samples.     

Inhibition of Topoisomerases by Fatty Acids

The inhibitory effects of various fatty acids on topoisomerases were examined, and their structure-activity relationships and mechanism of action were studied. Saturated fatty acids (C_6:0 to C_22:0) did not inhibit topoisomerase I, but cis-unsaturated fatty acids (C_16:1 to C_22:1) with one double bond showed strong inhibition of the enzyme. The inhibitory potency depended on the carbon chain length and the position of the double bond in the fatty acid molecule. The trans-isomer, methyl ester and hydroxyl derivative of oleic acid had no or little inhibitory effect on topoisomerases I and II. Among the compounds studied petroselinic acid and vaccenic acid (C_18:1) with a cis-double bond were the potent inhibitors. Petroselinic acid was a topoisomerase inhibitor of the cleavable complex-nonforming type and acted directly on the enzyme molecule in a noncompetitive manner without DNA intercalation.     

Essential‐Oil and Fatty‐Acid Composition,and Antioxidant Activity of Extracts of Ficaria kochii

The essential‐oil and fatty‐acid composition of the aerial parts of Ficaria kochii (Ledeb .) Iranshahr & Rech .f. native to Iran, and the antioxidant activity of various extracts of this plant were examined. The study by GC‐FID and GC/MS analysis of the essential oil resulted in the identification of 61 compounds, representing 86.01% of the total oil composition. Phytol (10.49%), farnesol (7.72%), methyl linoleate (5.57%), and α‐farnesene (4.96%) were the main components. The fatty‐acid composition of the aerial parts of F. kochii was also analyzed by GC/MS. The major components were palmitic acid (25.9%), linolenic acid (25.3%), and linoleic acid (17.5%). Polyunsaturated fatty acids (PUFAs) were found in higher amounts than saturated fatty acids. The possible antioxidant activity of various extracts (prepared by using solvents with different polarity) of the F. kochii aerial parts was evaluated by screening for their 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical‐scavenging activity, Fe^III‐reducing power, total antioxidant activity, and inhibitory activity in the linoleic acid‐peroxidation system. H₂O proved to be the most efficient solvent for the extraction of antioxidants, as the H₂O extract contained the highest amount of phenolic compounds (2.78±0.23 GAE/g dry matter) and also exhibited the strongest antioxidant capacity in all the assays used. The results of the present investigation demonstrated that the aerial parts of F. kochii can be used as natural and safe nutrition supplement in place of synthetic ones.     

An active site mutant of Escherichia coli cyclopropane fatty acid synthase forms new non-natural fatty acids providing insights on the mechanism of the enzymatic reaction

We have produced and purified an active site mutant of the Escherichia coli cyclopropane fatty acid synthase (CFAS) by replacing the strictly conserved G236 within cyclopropane synthases, by a glutamate residue, which corresponds to E146 of the homologous mycolic acid methyltransferase, Hma, producing hydroxymethyl mycolic acids. The G236E CFAS mutant had less than 1% of the in vitro activity of the wild type enzyme. We expressed the G236E CFAS mutant in an E. coli (DE3) strain in which the chromosomal cfa gene had been deleted. After extraction of phospholipids and conversion into the corresponding fatty acid methyl esters (FAMEs), we observed the formation of cyclopropanated FAMEs suggesting that the mutant retained some of the normal activity in vivo. However, we also observed the formation of new C17 methyl-branched unsaturated FAMEs whose structures were determined using GC/MS and NMR analyses. The double bond was located at different positions 8, 9 or 10, and the methyl group at position 10 or 9. Thus, this new FAMEs are likely arising from a 16:1 acyl chain of a phospholipid that had been transformed by the G236E CFAS mutant in vivo. The reaction catalyzed by this G236E CFAS mutant thus starts by the methylation of the unsaturated acyl chain at position 10 or 9 yielding a carbocation at position 9 or 10 respectively. It follows then two competing steps, a normal cyclopropanation or hydride shift/elimination events giving different combinations of alkenes. This study not only provides further evidence that cyclopropane synthases (CSs) form a carbocationic intermediate but also opens the way to CSs engineering for the synthesis of non-natural fatty acids.     

Phytanic acid alpha-oxidation,new insights into an old problem: a review

Phytanic acid (3,7,10,14-tetramethylhexadecanoic acid) is a branched-chain fatty acid which is known to accumulate in a number of different genetic diseases including Refsum disease. Due to the presence of a methyl-group at the 3-position, phytanic acid and other 3-methyl fatty acids can not undergo β-oxidation but are first subjected to fatty acid α-oxidation in which the terminal carboxyl-group is released as CO₂. The mechanism of α-oxidation has long remained obscure but has been resolved in recent years. Furthermore, peroxisomes have been found to play an indispensable role in fatty acid α-oxidation, and the complete α-oxidation machinery is probably localized in peroxisomes. This Review describes the current state of knowledge about fatty acid α-oxidation in mammals with particular emphasis on the mechanism involved and the enzymology of the pathway.     

Correlation analysis between fatty acid compositions of zooplankter individuals, fed on different phytoplankton species by means of pyrolysis–gas chromatography combined with on-line methylation

Pyrolysis–gas chromatography (Py–GC) combined with on-line methylation was applied to a correlation analysis between the distributions of fatty acid components in the lipids of zooplankter individuals and those of ingested algae using principal component analysis (PCA). Py–GC in the presence of organic alkali, tetramethylammonium hydroxide (TMAH), was used to estimate the apparent distributions of fatty acid components contained in a single individual zooplankter weighing several tens of micrograms and a small sample size of ingested algae samples in the order of 10 μg. The observed fatty acid compositions were used as a database for the PCA in order to discriminate the zooplankton and ingested algae samples. The result obtained indicated that the fatty acid compositions of zooplankton individuals used in this work were significantly reflected in those of their ingested food in spite of some contribution from isomerization and/or elongation of fatty acid components during digestion of the ingested algae phytoplankton in living zooplankters.     

Determination of the structure of the fatty acid chain in a cyclic lipopeptide using GC-MS

A GC-EIMS method to determine the structure of the fatty acid chains in cyclic lipopeptides is described. The structure of the fatty acid chains can be determined by the characteristic peaks of the MS spectrogram according to the fact that the alpha cleavage predominates the MS of a fatty acid with amino and hydroxy groups, while the McLafferty rearrangement predominates the MS of one without amino or hydroxy group. The characteristics of the strongest peaks of 103 and 102 in MS spectrograms due to alpha cleavage represent the beta-hydroxy-fatty acid and the beta-amino fatty acid, respectively; the strongest peak of 117 due to alpha cleavage and the relatively weak peak of 88 due to McLafferty rearrangement indicate the beta-hydroxy-fatty acid with a branched methyl group at its alpha position. The strongest peak of 74 due to McLafferty suggests the fatty acid without hydroxy or amino group. The ratio of relative intensity (I(43)/I(57)) characterizes the branches of alkyl chains. The greater I(43)/I(57) corresponds to an iso alkyl, and the smaller I(43)/I(57) corresponds to an anteiso alkyl. This method can be used to determine the full structure of the fatty acid chains in lipopeptides.     

Chemotaxonomic characterization of rice seedling blight complex using fatty acid methyl ester (FAME) profiles

Rice seedling blight is an important disease caused by a complex of fungi that include Fusarium, Rhizopus, Pythium, and Trichoderma species. A modified MIDI method was used for extraction of fatty acids from these causal pathogens, and fatty acid methyl ester (FAME) profiles were characterized. Factors that might affect fatty acid production, such as period of culture and saponification in extraction, were also evaluated. A total of 14 fatty acids were detected, and FAME profiles showed quantitative and qualitative variations by discriminant analysis and principal component analysis. Genus-specific FAME profiles consisting of the types of fatty acid produced and remarkable components of individual fatty acids were observed. The possibility of application as chemotaxonomic methods based on the FAME profiles for diagnosis of the rice seedling blight complex is also discussed.     

Trypanosoma cruzi: Fatty acid metabolism in vitro

Trypanosoma cruzi populations, composed primarily of trypomastigote forms, readily converted palmitic acid, linoleic acid, oleic acid, and stearic acid to CO₂. Appreciable amounts of carbon from these four fatty acids were also incorporated into neutral and phospholipid lipids by these parasites. Palmitic acid, a 16 carbon saturated fatty acid, was converted at rates greater than those of the other three fatty acids.     

Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids

Long-chain unsaturated fatty acids, such as linoleic acid, show antibacterial activity and are the key ingredients of antimicrobial food additives and some antibacterial herbs. However, the precise mechanism for this antimicrobial activity remains unclear. We found that linoleic acid inhibited bacterial enoyl-acyl carrier protein reductase (FabI), an essential component of bacterial fatty acid synthesis, which has served as a promising target for antibacterial drugs. Additional unsaturated fatty acids including palmitoleic acid, oleic acid, linolenic acid, and arachidonic acid also exhibited the inhibition of FabI. However, neither the saturated form (stearic acid) nor the methyl ester of linoleic acid inhibited FabI. These FabI-inhibitory activities of various fatty acids and their derivatives very well correlated with the inhibition of fatty acid biosynthesis using [(14)C] acetate incorporation assay, and importantly, also correlated with antibacterial activity. Furthermore, the supplementation with exogenous fatty acids reversed the antibacterial effect of linoleic acid, which showing that it target fatty acid synthesis. Our data demonstrate for the first time that the antibacterial action of unsaturated fatty acids is mediated by the inhibition of fatty acid synthesis.     

Mutants of Arabidopsis with alterations in seed lipid fatty acid composition

Summary A diverse collection of mutants of Arabidopsis with altered seed lipid compositions was isolated by determining the fatty acid composition of samples of seed from 3,000 mutagenized lines. A series of mutations was identified that caused deficiencies in the elongation of 181 to 201, desaturation of 181 to 182, and desaturation of 182 to 183. In each of these cases the wild type exhibited incomplete dominance over the mutant allele. These results, along with results from earlier studies, point to a major influence of gene dosage in determining the fatty acid composition of seed lipids. A mutation was also isolated that resulted in increased accumulation of 183. On the basis of the effects on fatty acid composition, the nature of the biochemical lesion in three of the mutants could be tentatively attributed to deficiencies in activities of specific enzymes. The other mutant classes had relatively less pronounced changes in fatty acid composition. These mutants may represent alterations in genes that regulate lipid metabolism or seed development. The availability of the mutants should provide new opportunities to investigate the mechanisms that control seed lipid fatty acid composition.Abbreviations FAMES (fatty acid methyl esters) - PC (phosphatidylcholine) - 181 (oleic acid) - 182 (linoleic acid) - 183 (linolenic acid) - 201 (eicosenoic acid) Supported in part by grants from the USDA (No. 89-37262-4388), USDA/NSF/DOE Plant Science Center Program, the U.S. Department of Energy (No. AC02-76ER01338), Karlshamns Research Foundation, and the WSU Research and Arts Committee     

Distribution of C22-, C24- and C26-α-Unit-Containing Mycolic Acid Homologues in Mycobacteria

There are three mycolic acid homologues with C₂₂-, C₂₄- and C₂₆-α-units in Mycobacterium. In order to reveal the composition and distribution of these homologues in each subclass and molecular species of mycolic acids and to compare them with the composition of constitutive non-polar fatty acids (free and bound forms), we have separated non-polar fatty acids and each subclass of mycolic acids from 21 mycobacterial species by thin-layer chromatography, and analyzed non-polar fatty acid methyl esters by gas chromatography (GC) and the cleavage products of methyl mycolate by pyrolysis GC. We further performed mass chromatographic analysis of trimethylsilyl (TMS) ether derivatives of mycolic acid methyl esters by monitoring [B-29]⁺ ions (loss of CHO from the α-branched-chain structure of mycolic acids) of m/z 426, 454 and 482 which are attributed to C₂₂-, C₂₄- and C₂₆-α-units of TMS ether derivatives of methyl mycolates, respectively, (Kaneda, K. et al, J. Clin. Microbiol. 24: 1060-1070, 1986). By pyrolysis GC, C_22:0, C_24:0 and C_26:0 fatty acid methyl esters generated by the C₂-C₃ cleavage of C₂₂-, C₂₄- and C₂₆-α-unit-containing mycolic acid methyl esters, respectively, were detected. Their proportion was almost the same among subclasses of mycolic acids in every Mycobacterium and also similar to the proportion of constitutive non-polar C_22:0, C_24:0 and C_26:0 fatty acids. By mass chromatography, the composition and distribution of C₂₂- and C₂₄-α-unit-containing homologues were revealed to be similar between α- and α'-mycolic acids in every Mycobacterium. We further analyzed in detail M. vaccae and demonstrated that the mass chromatogram of C₂₂-α-unit-containing homologue was analogous in shape to that of the C₂₄-α-unit-containing one, with the latter mass chromatogram being up-shifted from the former by two carbon numbers, in every subclass of α-, α'-, keto and dicarboxy mycolic acids. The present study suggests that the compositions of three homologues of both mycolic acids and constitutive non-polar fatty acids, which are characteristic to each mycobacterial species, may reflect the proportion of the amount of free C_22:0, C_24:0 and C_26:0 fatty acids synthesized in the cell. It is further demonstrated that intermolecular condensation of two fatty acids which become α- and β-units of mycolic acids will occur independently of the carbon chain length or kinds of polar moieties of fatty acids.     

A simplified and efficient method for the analysis of fatty acid methyl esters suitable for large clinical studies

Conventional sample preparation for fatty acid analysis is a complicated, multiple-step process, and gas chromatography (GC) analysis alone can require >1 h per sample to resolve fatty acid methyl esters (FAMEs). Fast GC analysis was adapted to human plasma FAME analysis using a modified polyethylene glycol column with smaller internal diameters, thinner stationary phase films, increased carrier gas linear velocity, and faster temperature ramping. Our results indicated that fast GC analyses were comparable to conventional GC in peak resolution. A conventional transesterification method based on Lepage and Roy was simplified to a one-step method with the elimination of the neutralization and centrifugation steps. A robotics-amenable method was also developed, with lower methylation temperatures and in an open-tube format using multiple reagent additions. The simplified methods produced results that were quantitatively similar and with similar coefficients of variation as compared with the original Lepage and Roy method. The present streamlined methodology is suitable for the direct fatty acid analysis of human plasma, is appropriate for research studies, and will facilitate large clinical trials and make possible population studies.     

Fatty acid binding proteins from developing human fetal brain: Characterization and binding properties

Two fatty acid binding proteins (FABPs) of identicalM _r, 13 kDa, have been isolated from developing human fetal brain. A delipidated 105,000 g supernatant was incubated with [1 -¹⁴C]oleate and subjected to a Sephacryl S-200 column followed by gel filtration chromatography on a Sephadex G-75 column and ion-exchange chromatography using a DEAE-Sephacel column. Purity was checked by UV spectroscopy, SDS-PAGE, isoelectric focusing and immunological cross-reactivity. The two FABPs designated as DE-I (pI 5.4) and DE-II (pI 6.9) showed cross-reactivity with each other and no alteration at the antigenic site during intrauterine development. Anti-human fetal brain FABP does not cross-react with purified human fetal heart, gut, lung or liver FABPs. The molecular mass of DE-I and DE-II is lower than those of fetal lung and liver FABPs. Like liver FABP, these proteins bind organic anions, fatty acids and acyl CoAs but differ in their binding affinities. Both DE-I and DE-II have been found to exhibit higher affinity for oleate (K _d = 0.23 μM) than palmitate (K _d = 0.9μM) or palmitoyl-CoA (K _d = 0.96 μM), with DE-I binding less fatty acids than DE-II. DE-II is more efficient in transferring fatty acid from phospholipid vesjcles than DE-I indicating that human fetal brain FABPs may play a significant role in fatty acid transport in developing fetal brain.     

Isolation and characterization of fatty acid methyl ester (FAME)‐producing Streptomyces sp. S161 from sheep (Ovis aries) faeces

An actinomycete producing oil‐like mixtures was isolated and characterized. The strain was isolated from sheep faeces and identified as Streptomyces sp. S161 based on 16S rRNA gene sequence analysis. The strain showed cellulase and xylanase activities. The ¹H nuclear magnetic resonance (NMR) spectra of the mixtures showed that the mixtures were composed of fatty acid methyl esters (52·5), triglycerides (13·7) and monoglycerides (9·1) (mol.%). Based on the gas chromatography–mass spectrometry (GC‐MS) analysis, the fatty acid methyl esters were mainly composed of C14‐C16 long‐chain fatty acids. The results indicated that Streptomyces sp. S161 could produce fatty acid methyl esters (FAME) directly from starch. To our knowledge, this is the first isolated strain that can produce biodiesel (FAME) directly from starch.

Significance and Impact of the Study

Nowadays, production of biodiesel is based on plant oils, animal fats, algal oils and microbial oils. Lipid mostly consists of triacylglycerols (TAG), and conversion of these lipids into fatty acid short‐chain alcohol esters (methanol or ethanol) is the final step in biodiesel production. In this study, an oil‐producing Streptomyces strain was isolated from sheep faeces. The oil was composed of C₁₄‐C₁₆ long‐chain fatty acid methyl esters, triglycerides and monoglycerides. This is the first isolated strain‐producing biodiesel (FAME) directly from starch. Due to showing cellulase and xylanase activities, the strain would be helpful for converting renewable lignocellulose into biodiesel directly.     

Glucose stimulates a decrease of the fatty acid saturation degree in Acinetobacter calcoaceticus

The fatty acid composition of Acinetobacter calcoaceticus 69-V was determined under various growth conditions. Saturated, unsaturated, and hydroxy fatty acids with chain lengths of 12–18 carbon atoms predominated in the fatty acid profile. With acetate or propanol as growth substrates, the ratio of saturated to unsaturated fatty acids varied with changes in the temperature. This was the only adaptive mechanism detected that compensated for the physical effects of temperature alterations on the cell membranes. The fatty acid composition of A. calcoaceticus grown at 40 °C had a saturation degree of approximately 50%; after growth at 20 °C it was approximately 35%. In the presence of a carbon and energy source, A. calcoaceticus was able to respond to temperature reductions under oxic conditions regardless of whether fatty acid biosynthesis was inhibited or not. This suggests an aerobic mechanism of fatty acid biosynthesis and the involvement of a fatty acid desaturase system. Addition of the non-growth substrate, glucose, helped the organism to adapt to lower temperature. The molecular mechanism of the aid is not really understood. The oxidation of glucose could provide the desaturase either with electrons directly via a pyrrolo-quinoline-quinone-linked glucose dehydrogenase or with NADH after fatty acid degradation has been initiated by ATP generated by the oxidation of glucose. Received: 19 June 1998 / Accepted: 28 December 1998