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.     

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.     

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.     

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.     

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.     

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.     

New estimation method for fatty acid composition in oil using near infrared spectroscopy

The absorption bands of cis-unsaturation and the carbon chain length of the fatty acid moieties in oil appear in the near infrared (NIR) wavelength region, especially around 1600-1800 nm. Using this region, a new estimation method for fatty acid composition analysis is proposed. Because the differences of the original NIR spectra are miniscule even in this region, the second derivative NIR spectra were examined in order to estimate the fatty acid composition in oil exclusively from the spectral patterns obtained. The parameters for calculating the second derivative NIR spectra were examined to make the spectral difference clearer. In any parameter, the absorption band was shifted to the shorter wavelength region when the unsaturation in fatty acid moieties increased, and it was shifted to the longer wavelength region when the carbon chain length increased. When the parameters were correct, this NIR method can estimate the fatty acid composition roughly, but simply, easily, and sometimes nondestructively.     

Identification of bacteria from single colonies by fatty acid analysis.

While a commercially available system for microbial identification by fatty acid analysis (Microbial Identification System, MIDI, Newark, DE, USA) is available, it requires approximately 40-mg wet weight of biomass. Various authors have published methods for fatty acid analysis of single colonies, but no database of organisms has been developed for those methods. A modification of the MIDI system to increase sensitivity while maintaining relative retention times and peak areas allows the standard peak naming tables and libraries of organisms to be used with single colonies. Samples are evaporated down before injection to concentrate the fatty acids, while splitless injection is used to allow more sample to enter the gas chromatograph. Several known bacteria were correctly identified by this method.     

Antimicrobial activity of fatty acid methyl esters of some members of Chenopodiaceae

Fatty acid methyl ester (FAME) extracts of four halophytic plants, viz. Arthrocnemum indicum, Salicornia brachiata, Suaeda maritima and Suaeda monoica belonging to the family Chenopodiaceae, were prepared and their composition was analyzed by GC-MS. The FAME extracts were also screened for antibacterial and antifungal activities. The GC-MS analysis revealed the presence of more saturated fatty acids than unsaturated fatty acids. Among the fatty acids analyzed, the relative percentage of lauric acid was high in S. brachiata (61.85%). The FAME extract of S. brachiata showed the highest antibacterial and antifungal activities among the extracts tested. The other three extracts showed potent antibacterial and moderate anticandidal activities.     

Double bond localization in minor homoallylic fatty acid methyl esters using acetonitrile chemical ionization tandem mass spectrometry

Double bond position in natural fatty acids is critical to biochemical properties, however, common instrument-based methods cannot locate double bonds in fatty acid methyl esters (FAME), the predominant analysis form of fatty acids. A recently described mass spectrometry (MS) method for locating double bonds in FAME is reported here for the analysis of minor (<1%) components of real FAME mixtures derived from three natural sources; golden algae (Schizochytrium sp.), primate brain white matter, and transgenic mouse liver. Acetonitrile chemical ionization tandem MS was used to determine double bond positions in 39 FAME, most at concentrations well below 1% of all fatty acid methyl esters. FAME identified in golden algae are 14:1n-6, 14:3n-3, 16:1n-7, 16:2n-6, 16:3n-6, 16:3n-3, 16:4n-3, 18:2n-7, 18:3n-7, 18:3n-8, 18:4n-3, 18:4n-5, 20:3n-7, 20:4n-3, 20:4n-5, 20:4n-7, 20:5n-3, and 22:4n-9. Additional FAME identified in primate brain white matter are 20:1n-7, 20:1n-9, 20:2n-7, 20:2n-9, 22:1n-7, 22:1n-9, 22:1n-13, 22:2n-6, 22:2n-7, 22:2n-9, 22:3n-6, 22:3n-7, 22:3n-9, 22:4n-6, 24:1n-7, 24:1n-9, and 24:4n-6. Additional FAME identified in mouse liver are 26:5n-6, 26:6n-3, 28:5n-6, and 28:6n-3. The primate brain 22:3n-7 and algae 18:4n-5 are novel fatty acids. These results demonstrate the usefulness of the technique for analysis of real samples. Tables are presented to aid in interpretation of acetonitrile CIMS/MS spectra.     

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.     

Steroidal fatty acid esters

Several years ago we discovered an unexpected family of steroidal metabolites, steroidal fatty acid esters. We found that fatty acid esters of 5-ene-3β-hydroxysteroids, pregnenolone and dehydroisoandrosterone are present in the adrenal. Subsequently, others have shown the existence of these non-polar 5-ene-3β-hydroxysteroidal esters in blood, brain and ovaries. Currently, almost every family of steroid hormone is known to occur in esterified form. We have studied the esters of the estrogens and glucocorticoids in some detail, and have found that these two steroidal families are esterified by separate enzymes. In a biosynthetic experiment performed simultaneously with estrodiol and corticosterone, we established that the fatty acid composition of the steroidal esters is quite different. The corticoid is composed predominantly of one fatty acid, oleate, while the estradiol esters are extremely heterogeneous. Our studies have demonstrated that the estrogens are extremely long-lived hormones, that they are protected by the fatty acid from metabolism. They are extremely potent estrogens, with prolonged activity. Esterification appears to be the only form of metabolism that does not deactivate the biological effects of estradiol. We have demonstrated the biosynthesis of fatty acid esters of estriol, monoesters at both C-16 and C-17β. They too are very potent estrogens. These fatty acid esters of the estrogens are the endogenous analogs of estrogen esters, like benzoate, cypionate, etc., which have been used for decades, pharmacologically because of their prolonged therapeutic potency. We have found that the estradiol esters are located predominantly in hydrophobic tissues, such as fat. Sequestered in these tissues, they are an obvious reservoir of estrogenic reserve, requiring only an esterase for activation. To the contrary the biological activity of the fatty acid esters of the glucocorticoid, corticosterone, is not different from that of its free parent steroid. We have shown that the rapid kinetics of its induction of gluconeogenic responses is caused by its labile C-21 ester which is rapidly hydrolyzed by esterase enzymes. While it appears that the physiological role of the estrogen esters may be related to their long-lived hormonal activity, the role of the other families of steroidal esters is not yet apparent. They, and perhaps the estrogen esters as well, must serve other purposes. Indeed they may serve important biological functions beyond those which we ordinarily associate with steroid hormones.     

A monolithic lipase reactor for biodiesel production by transesterification of triacylglycerides into fatty acid methyl esters

An enzymatic reactor with lipase immobilized on a monolithic polymer support has been prepared and used to catalyze the transesterification of triacylglycerides into the fatty acid methyl esters commonly used for biodiesel. A design of experiments procedure was used to optimize the monolithic reactor with variables including control of the surface polarity of the monolith via variations in the length of the hydrocarbon chain in alkyl methacrylate monomer, time of grafting of 1-vinyl-4,4-dimethylazlactone used to activate the monolith, and time used for the immobilization of porcine lipase. Optimal conditions involved the use of a poly(stearyl methacrylate-co-ethylene dimethacrylate) monolith, grafted first with vinylazlactone, then treated with lipase for 2 h to carry out the immobilization of the enzyme. Best conditions for the transesterification of glyceryl tributyrate included a temperature of 37°C and a 10 min residence time of the substrate in the bioreactor. The reactor did not lose its activity even after pumping through it a solution of substrate equaling 1,000 reactor volumes. This enzymatic reactor was also used for the transesterification of triacylglycerides from soybean oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel.