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.     

Two-column gas-liquid chromatography of fatty acid methyl esters

Fatty acid methyl esters were separated into fractions according to chain length on a nonpolar gas-liquid chromatographic column. These fractions were collected and rechromatographed on a polar column. Temperature programming was used in both cases. Data are given for the accuracy of the double procedure applied to a synthetic mixture.     

Preparation of fatty acid methyl esters for gas-liquid chromatography

A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45°C overnight or heated at 100°C for 1–1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be <1.4%. The yields of FAMEs were >96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC.     

Permeability behavior of liposomes prepared from fatty acids and fatty acid methyl esters

The permeability properties of liposomes prepared at pH 8.7 from a fatty acid and either methyl oleate or methyl elaidate, with or without cholesterol, were investigated. The fatty acids used were oleic acid, elaidic acid, and the selenium-containing fatty acids 9-selenaheptadecanoic acid and 13-selenaheneicosanoic acid. The liposomes trapped sucrose and carboxyfluorescein. Their volume change resulting from osmotic shock was directly proportional to the change in absorbance (light scattering). Liposomes prepared from oleic acid and either methyl oleate or methyl elaidate underwent osmotic swelling much more slowly than liposomes prepared from elaidic acid and either methyl oleate or methyl elaidate. Incorporation of cholesterol decreased the initial rate of erythritol permeation, especially in liposomes containing methyl oleate. The swelling rates of liposomes prepared with the selenium-containing fatty acids indicated that incorporation of methyl elaidate gave more tightly packed bilayers than did incorporation of methyl oleate. The effect of cholesterol on the initial rate of erythritol influx was greater in oleic acid and elaidic acid liposomes than in selenium-containing fatty acid liposomes, indicating that the large bulk of the selenium heteroatom suppresses the ability of cholesterol to interact with the hydrocarbon chain.     

Identification of foodborne bacteria by infrared spectroscopy using cellular fatty acid methyl esters

Identification of bacterial species by profiling fatty acid methyl esters (FAMEs) has commonly been carried out by using a 20-min capillary gas chromatographic procedure followed by library matching of FAME profiles using commercial MIDI databases and proprietary pattern recognition software. Fast GC (5 min) FAME procedures and mass spectrometric methodologies that require no lipid separation have also been reported. In this study, bacterial identification based on the rapid (2 min) infrared measurement of FAME mixtures was demonstrated. The microorganisms investigated included Gram positive bacteria Staphylococcus aureus, Listeria monocytogenes, Bacillus anthracis, and Bacillus cereus, and Gram negative bacteria from the family Enterobacteriacae: Yersinia enterocolitica, Salmonella typhimurium, Shigella sonnei, and Escherichia coli (four strains of E. coli), and non-Enterobacteriacae: Vibrio cholerae, Vibrio vulnificus, and Vibrio parahemolyticus. Foodborne bacterial mixtures of FAMEs were measured by using an attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopic procedure and discriminated by multivariate analysis. Results showed that the Enterobacteriacae could be discriminated from the vibrios. The identification was at the level of species (for the Bacillus and Vibrio genera) or strains (for the E. coli species). A series of bacterial FAME test samples were prepared and analyzed for accuracy of identification, and all were correctly identified. Our results suggest that this infrared strategy could be used to identify foodborne pathogens.     

Determination of the specific radioactivity of fatty acids separated as their methyl esters by gas-liquid chromatography

Free or combined (3)H-labeled fatty acids are converted to their methyl-(14)C esters or, if labeled with (14)C, to their methyl-(3)H esters. For a given specific radioactivity of the methyl group, the nuclide ratio in the esters separated by GLC is a direct measure of the specific radioactivity of the fatty acids, and quantitative collection is unnecessary. Methods of methylation with minimum quantities of labeled methanol, and of deriving nuclide ratios from channel ratios in a scintillation spectrometer, are given.     

Gas chromatographic-mass spectrometric detection of 2- and 3-hydroxy fatty acids as methyl esters from soil,sediment and biofilm

Hydroxy fatty acids (OH-FAs) can be used in the characterization of microbial communities, especially Gram-negative bacteria. We prepared methyl esters of 2- and 3-OH-FAs from the lipid extraction residue of soil, sediment, and biofilm samples without further purification or derivatization of hydroxyl groups. OH-FA methyl esters were analyzed using a gas chromatograph equipped with a mass selective detector (GC-MS). The ions followed in MS were m/z 103 for 3-OH-FAs and m/z 90 and M-59 for 2-OH-FAs. The rapid determination of 3- and 2-OH-FAs concomitantly with phospholipid fatty acids provided more detailed information on the microbial communities present in soil, sediment, and drinking water biofilm.     

Fatty acid analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas chromatography

A method to prepare fatty acid methyl esters was developed for fatty acid analysis of triacylglycerols by gas chromatography (GC). Triacylglycerols were mixed with methanolic CH₃ONa in hexane containing a mid-polar solvent for 10 s at room temperature. Under these conditions, trioleoylglycerol was converted to methyl oleate with an average yield of 99.3%. This procedure gave reliable and reproducible data on fatty acid compositions determined by GC.     

Uptake of long-chain fatty acid methyl esters by mammalian cells

Albumin-bound long-chain fatty acid methyl esters (ME) were taken up and utilized by Ehrlich ascites tumor cells and slices of rat heart, liver, and kidney. Much more ME than albumin was taken up by the tumor cells, indicating that ME dissociated from the carrier protein during their uptake. 70-80% of the radioactivity associated with the cells after 1 min of incubation at 37 degrees C remained as ME. The results of studies with metabolic inhibitors and glucose suggest that uptake of ME is an energy-independent process. Changes in incubation medium pH between 7.8 and 6.5 did not markedly alter uptake of ME. Cells incubated with FFA and methanol did not synthesize ME. These findings indicate that ME are taken up intact, and they suggest that the presence of an anionic carboxyl group is not essential for the binding of a long-chain aliphatic hydrocarbon to a mammalian cell. When incubation with labeled ME was continued for 1 hr, increasing amounts of radioactivity were recovered in FFA, phospholipids, neutral lipid esters, and CO(2). ME radioactivity associated with the cells after a brief initial incubation was released in the form of ME and FFA when the cells were incubated subsequently in a medium containing albumin. If the second incubation medium contained no albumin, most of the ME radioactivity initially associated with the cells was incorporated into phospholipids, neutral lipid esters, and CO(2). These results suggest that much of the ME which is taken up, is hydrolyzed to FFA, and that the fatty acids derived from ME are available for further metabolism.     

Dimeric epoxy fatty acid methyl esters: formation,chromatography and mass spectrometry

The formation of dimers is reported from the thermal treatment of a series of epoxy fatty acid methyl esters. These compounds were isolated from the reaction mixture by steric exclusion chromatography and were subsequently characterised by their high resolution electron impact and ammonia chemical ionisation mass spectra. The spectra were consistent in each case with the presence of a mixture of four possible positional isomers each containing an ether bridge linking a pair of fatty acid methyl esters across the carbon chains, with a keto group on a carbon adjacent to the bridge on one of the esters.     

The anticlastogenic potential of fatty acid methyl esters

To test for possible anticlastogenic effects of fatty acids, the methyl esters of fatty acids--short-chain to long-chain--were examined on busulfan in Chinese hamster bone-marrow cells using the chromosome aberration test. When the experimental animals were treated with fatty acid esters and the mutagen, the chromosome-breaking actions of busulfan were not modulated by the short-chain fatty acids, but the fatty acids from lauric acid (C12) up to nonadecanoic acid (C19) reduced the rate of aberrant metaphases from 9.4 to about 3% at doses of 100 mg/kg and less. Other chemical properties of the fatty acids (saturated or not, number of double bonds, even- or odd-numbered) had no influence on the anticlastogenic effects. The only exceptions to this rule were arachidonic acid, which had no effect, and gamma-linolenic acid, which had no consistent effect on the action of busulfan.     

Stability of unsaturated methyl esters of fatty acids on surfaces

The stability of unsaturated methyl esters is greater when they are adsorbed on silica gel than when a glass surface is used. Storage of small samples adsorbed on silica gel may be a convenient addition to conventional methods of protecting labile fats against autoxidation.     

Biotransformation of free fatty acids in mixtures to methyl ketones by Monascus purpureus

Summary Monascus purpureus converts short-chain fatty acids to methyl ketones. The regulation of the metabolic pathway is similar to that found in Penicillium roquefortii. There are differences in the actual amount of precursors metabolized. The fermentation of fatty acid mixtures led to methyl ketone mixtures. The metabolism of each fatty acid was dependent on the precursor composition. Offprint requests to: C. Kranz     

Inhibition of oxidation of unsaturated fatty acid methyl esters by essential oils

The essential oils from 16 various spice plants were studied as natural antioxidants for the inhibition of autooxidation of polyunsaturated fatty acids methyl esters isolated from linseed oil. The content of methyl oleate, methyl linoleate, and methyl linolenoate after 1, 2, and 4 months of autooxidation were used as criteria to estimate the antioxidant efficiencies of essential oils. In 4 months, 92% of the methyl linolenoate and 79% of the methyl linoleate were oxidized in a control sample of a model system. It was found that the most effective antioxidants were essential oils from clove bud, cinnamon leaves, and oregano. They inhibited autooxidation of methyl linolenoate by 76–85%. The antioxidant properties of these essential oils were due to phenols— eugenol, carvacrol, and thymol. Essential oil from coriander did not contain phenols, but it inhibited methyl linolenoate oxidation by 38%. Essential oils from thyme, savory, mace, lemon, and tea tree inhibited methyl linolenoate oxidation by 17–24%. The other essential oils had no antioxidant properties.