Estimating the average carbon chain length of saturated fatty acid esters by infrared spectroscopy

The average carbon chain length of saturated fatty acid esters can be determined by comparing absorption intensities in the 3.3 and 5.75 μ infrared regions. Data are presented for triglycerides, monoglycerides, and methyl esters. The method was used to follow the fractionation of hydrogenated milk fat from acetone, and the average values for fatty acid chain length were in good agreement with those obtained by gas chromatographic methyl ester analysis. The I.R. method is particularly applicable when only a few mg of sample is available and the material being fractionated contains both long and short chain saturated fatty acids. Authorized for publication August 9, 1961, as Paper No. 2590 in the Journal Series of the Pennsylvania Agricultural Experiment Station. Supported in part by the U. S. Public Health Service (H3632).     

Separation of saturated/unsaturated fatty acids

Fatty acid mixtures can be separated into one fraction rich in saturated fatty acids and the other rich in unsaturated acids. Since saturated fatty acids have a higher melting point than unsaturated, liquid mixture to be fractionated is cooled to a temperature at which the larger part of the saturated acids crystallize, while the greater part of unsaturated acids remain in liquid form. Different industrial methods to separate the two phases are described. The oldest and simplest method is slowly to cool and crystallize the mixture in shallow pans to form cakes which then are pressed in presses of different design. By applying high pressure, the liquid olein is thus squeezed out from the cake, leaving the stearin fraction behind. A new process to separate the phases is to mix an aqueous solution, containing a wetting agent, with the crystallized fatty acid mixture. The stearin crystals are thus wetted and transferred into the aqueous phase, which then can be separated from the olein phase in a centrifuge. The stearin/aqueous suspension is heated to melt the stearin, which can then be separated in a second centrifuge. Other methods to improve phase separation use organic solvents, among which are methanol, acetone, methyl formate and propane. In the solvent fraction process, the miscella has to be cooled to a lower temperature than in the aforementioned methods, due to the solubility effect of the solvents. The solvents are removed by distillation from the fraction. Typical operation results with different types of raw materials are given. The advantages and disadvantages of the different methods are discussed.     

Quantitative structure-property relationships for normal saturated and unsaturated fatty acids

Quantitative structure-property relationships, a technique of deriving properties of compounds from knowledge of their structures, has been applied to aliphatic carboxylic acids, an industrially important group of compounds. Equations have been developed from the molecular connectivity of the first order and the molar refraction as inputs which, on testing with data of seven properties, yielded average absolute deviations ranging between 0.9 and 6.7%. The compounds studied ranged between C₁ and C₃₀ for saturated and C₃ to C₂₂ for unsaturated fatty acids. The properties studied are normal melting and boiling points, critical temperature and pressure, and heats of fusion, combustion and formation.     

Esterification rates of some saturated and unsaturated fatty acids with glycerol

Esterification rates of eight commonly occurring fatty acids were studied at 180C using equivalent and equimolar amounts of glycerol with and without a cosolvent. The esterification with equivalent amounts of glycerol without cosolvent followed second order kinetics and proceeded at a similar rate for all acids examined. Esterifications with equimolar amounts of glycerol were kinetically complex and their speed depended on the solubility of glycerol in individual fatty acids.     

Comparison of radiolytic compounds from saturated and unsaturated triglycerides and fatty acids

Formation of certain radiolysis products from palmitic acid, oleic acid, tripalmitin and triolein has provided a means for comparing the radiolytic effects in saturated and unsaturated triglycerides and fatty acids. These substances were chosen to represent the major constituents of fat found in beef. Fractionation and concentration of radiolytic compounds from the irradiated samples was accomplished by the means of size exclusion chromatography. Quantitative and qualitative analyses were performed using a combined GC/MS computer system. In addition to the primary radiolytic compounds, recombination products of relatively high molecular weight and various propanediol diesters from the corresponding glyceryl moities were identified. Quantitative analyses indicated a greater yield of various radiolytic compounds from free fatty acids than from the corresponding triglycerides. Similarly, radiolytic compounds were produced in greater quantities from the saturated fats than the unsaturated fats. Most of the radiolytic compounds identified in this study have not been previously reported.     

Wide-line NMR spectra of some saturated and unsaturated long chain fatty acids

Wide-line NMR spectra were obtained on a series of homologous normal long chain fatty acids: decanoic, lauric, myristic, palmitic, stearic and behenic as well as three isostructural unsaturated acids: elaidic,trans-5-eicosenoic and brassidic. Also included are NMR spectra of metastable forms of both the saturated and unsaturated acids. NMR parameters are correlated with carbon chain length, crystal long spacing and density. Polymorphic forms are distinguished on the basis of line width and second moment differences. Presented in part at the Meeting of the Society for Applied Spectroscopy, October, 1970, New Orleans, La. So. Market. Nutr. Res. Div., ARS, USDA.     

Preparation and properties of oxalic acid salts of C18 saturated and unsaturated fatty amides

Crystalline oxalic acid salts of stearamide, oleamide and elaidamide consisting of 2 mol amide to 1 mol oxalic acid have been prepared and characterized by melting point and IR and X-ray diffraction measurements. Their IR spectra are compared to those of the amides. Long spacings are reported for the crystalline salts, and both long and short spacings for these amides and linoleamide. ARS, USDA     

JAOCS News Feature biosynthesis of saturated and unsaturated fatty acids by maturingcarthamus tinctorius L. seeds

Recent advances in the biosynthesis of fatty acids by maturing safflower seeds are described. We now can define the systems responsible for (a) the conversion of CO₂ to sucrose to acetyl CoA; (b) the condensation of acetyl CoA via the de novo acyl carrier protein pathway to the terminal product, palmityl acyl carrier protein; (c) the elongation of palmityl acyl carrier protein to stearyl acyl carrier protein by a specific malonyl acyl carrier proteinelongation system; (d) the desaturation of stearyl acyl carrier protein to oleic acid by a highly specific, soluble stearyl acyl carrier protein desaturase; (e) the further desaturation of oleyl CoA to linoleyl CoA; and (f) the transfer of acyl CoAs to glycerol-3-phosphate to the final product, a triacylglycerol and its coalescence to the typical high lipid containing mature seed, engorged with oil droplets. Future research should be directed to a fuller understanding of how the maturing seed triggers these complicated series of reactions, how all these enzymes are coordinated to allow a smooth flow of substrates for the formation of oil droplets, and, finally, how biochemical engineering can further improve oil crops in the ever growing demand for high yielding systems. Winner of the Award in Lipid Chemistry presented at the 48th Annual Fall Meeting, September, 1974, Philadelphia.     

Determination of unsaturated fatty acid composition by high-resolution nuclear magnetic resonance spectroscopy

High-resolution nuclear magnetic resonance (NMR) spectroscopy provides useful data for analyzing fatty acid compositions of edible vegetable oils. Quantitation of each fatty acid was carried out by evaluation of particular peaks. According to the ¹H NMR method, terminal methyl protons, divinyl protons, and allyl protons are useful to calculate linolenic acid, linoleic acid, and oleic acid, respectively. The ω-2 carbon, divinyl carbon, and allylic carbons were used for calculation of these acids by the ¹³C NMR method. Compositional results obtained by NMR coincided well with those of the conventional gas chromatography (GC) method. Results from ¹³C NMR were in better agreement with those from GC than were the results obtained by the ¹H NMR method.     

Determination of unsaturated fatty acid composition by high-resolution nuclear magnetic resonance spectroscopy

High-resolution nuclear magnetic resonance (NMR) spectroscopy provides useful data for analyzing fatty acid compositions of edible vegetable oils. Quantitation of each fatty acid was carried out by evaluation of particular peaks. According to the ¹H NMR method, terminal methyl protons, divinyl protons, and allyl protons are useful to calculate linolenic acid, linoleic acid, and oleic acid, respectively. The ω-2 carbon, divinyl carbon, and allylic carbons were used for calculation of these acids by the ¹³C NMR method. Compositional results obtained by NMR coincided well with those of the conventional gas chromatography (GC) method. Results from ¹³C NMR were in better agreement with those from GC than were the results obtained by the ¹H NMR method.     

Different metabolism of saturated and unsaturated long chain plasma free fatty acids by intestinal mucosa of rats

During fat absorption, unsaturated long chain fatty acids are esterified at a higher rate than saturated fatty acids of similar chain length. This phenomenon has been attributed to differences in the binding affinity of fatty acids to a cytosolic fatty acid-binding protein. As intestinal mucosa utilizes plasma free fatty acids as well, we investigated whether long chainplasma free fatty acids of different degree of saturation are metabolized also at different rates.³H-Palmitic and¹⁴C-linoleic acid complexed to rat serum were injected rapidly into a tail vein of fasting rats. One, 2 and 4 min later there was no difference between³H and¹⁴C-radioactivity in intestinal mucosa, suggesting equal initial uptake of the two labeled fatty acids from plasma. Despite their equal uptake, the incorporation of the isotopes into ester lipids was significantly different, however: at 2 min, 53.1±3.9% of³H and 73.8±4.6% of¹⁴C were recovered in ester lipids. Phospholipids and triglycerides accounted for most of the mucosal³H and¹⁴C. At 4 min, a similar distribution of isotopes in intestinal mucosal metabolites was found. These data show that despite equal initial uptake by intestinal mucosa unsaturated long chain fatty acids taken up from plasma are esterified to a higher and oxidized to a lower extent than saturated plasma free fatty acids. Unsaturated plasma free fatty acids, therefore, may provide a more important source of fatty acids for endogenous intestinal lipoprotein lipids than saturated plasma free fatty acids. It is speculated that the fatty acid binding protein might be operative not only in the intracellular transport and metabolism of luminal fatty acids but of plasma free fatty acids as well.     

Accumulation of neutral lipids by human skin fibroblasts: Differential effects of saturated and unsaturated fatty acids

The accumulation of neutral lipids by human skin fibroblasts grown in medium supplemented with fatty acids has been investigated. GM-10 cells incorporated exogenous fatty acids into both phospholipids and neutral lipids. More [¹⁴C] oleate, linoleate, or linolenate was incorporated into triacylglycerol than was [¹⁴C] palmitate or stearate. Supplementation of medium containing delipidized serum with unsaturated fatty acids resulted in far more stimulation of [¹⁴C] glycerol incorporation into triacylglycerol than did supplementation with saturated fatty acids. Palmitate- and stearate-fed cells incorporated sizable amounts of [¹⁴C] fatty acids and [¹⁴C] glycerol into diacylglycerol as well as triacylglycerol, especially at higher fatty acid concentrations. Increased oleate supplementation from 10–300 μM resulted in increased triacylglycerol synthesis and accumulation of discrete cytoplasmic lipid droplets; palmitate concentrations above 70 μm were toxic. Micrographs of the palmitate-fed cells showed electron translucent slits, suggesting solid depositions of saturated fat, rather than the discrete osmiophilic droplets found in oleate-fed cells. Although GM-10 cells can synthesize fully saturated triacylglycerols, these data suggest that in cells fed saturated fatty acids, solid depositions of neutral lipids may sequester diacylglycerols and thus limit triacylglycerol synthesis.     

Incorporation and distribution of saturated and unsaturated fatty acids into nuclear lipids of hepatic cells

Liver nuclear incorporation of stearic (18∶0), linoleic (18∶2n−6), and arachidonic (20∶4n−6) acids was studied by incubation in vitro of the [1-¹⁴C] fatty acids with nuclei, with or without the cytosol fraction at different times. The [1-¹⁴C] fatty acids were incorporated into the nuclei as free fatty acids in the following order: 18∶0>20∶4n−6≫18∶2n−6, and esterified into nuclear lipids by an acyl-CoA pathway. All [1-¹⁴C] fatty acids were esterified mainly to phospholipids and triacylglycerols and in a minor proportion to diacylglycerols. Only [1-¹⁴C] 18∶2n−6-CoA was incorporated into cholesterol esters. The incorporation was not modified by cytosol addition. The incorporation of 20∶4n−6 into nuclear phosphatidylcholine (PC) pools was also studied by incubation of liver nuclei in vitro with [1-¹⁴C]20∶4n−6-CoA, and nuclear labeled PC molecular species were determined. From the 15 PC nuclear molecular species determined, five were labeled with [1-¹⁴C]20∶4n−6-CoA: 18∶0–20∶4, 16∶0–20∶4, 18∶1–20∶4, 18∶2–20∶4, and 20∶4–20∶4. The highest specific radioactivity was found in 20∶4–20∶4 PC, which is a minor species. In conclusion, liver cell nuclei possess the necessary enzymes to incorporate exogenous saturated and unsaturated fatty acids into lipids by an acyl-CoA pathway, showing specificity for each fatty acid. Liver cell nuclei also utilize exogenous 20∶4n−6-CoA to synthesize the major molecular species of PC with 20∶4n−6 at the sn-2 position. However, the most actively synthesized is 20∶4–20∶4 PC, which is a quantitatively minor component. The labeling pattern of 20∶4–20∶4 PC would indicate that this molecular species is synthesized mainly by the de novo pathway.     

The fungicidal activity of the unsaturated fatty acids and quaternary salts prepared from fish oils

Summary Saturated and unsaturated fatty acids and 10 unsaturated fatty acid fractions and ethyl esters of unsaturated fatty acid fractions prepared from fish oils were tested on their inhibitory activity againstCandida albicans. Oxidation of highly unsaturated fractions from fish oil and ethyl esters of unsaturated fatty acid fractions of menhaden, pilchard, and cod liver oils increases their antifungal activity. Saturated and unsaturated quaternaries were tested for their antifungal activity. Hexadecyltriethylammonium bromide and hexadecylpyridinium bromide showed the highest activity againstCandida albicans, Aspergillus niger, andRhyzopus nigricans. Any lengthening of the carbon chain more than C₁₆ weakened the activity of both saturated triethylammonium bromide and pyridinium bromide. An increase of unsaturation enhanced it. The antifungal activity of quaternaries prepared from fish oils was about 4,000 times stronger than that of oxidized highly unsaturated fatty acid fractions prepared from fish oils. The decisive factor in the highly inhibitory activity of quaternaries against fungi might depend on their positively charged portion since the surface of microorganisms is, as a rule, negatively charged. Aided by a grant from the Collett-Week Company, Ossining, N. Y.     

Determination of structure of unsaturated fatty acids via reductive ozonolysis

An improved method of reductive ozonolysis for the determination of structure of unsaturated fatty acids is reported. The ozonization is carried out at −60–−70C by adding the sample dissolved in pentane to a .02–.03 M pentane solution of ozone. The reduction is effected by the Lindlar catalyst at 0C in pentane or in other solvents, such as the methyl esters of short-chain fatty acids or in dimethyl phthalate, and the aldehydic fragments are analyzed by gas-liquid chromatography (GLC). The method is applied to the structural analyses of methyl esters of oleic, linoleic, linolenic, and arachidonic acids. The sensitivity of the method is demonstrated by the analysis of methyl oleate containing a small amount of added methyl linolenate. Mixtures of methyl oleate and petroselinate are analyzed to demonstrate the identification of the simple aldehydes and ester-aldehydes, and to show the potential of the method for quantitative analysis of mixtures of unsaturated esters. Supported in part by the U.S. Public Health Service, N.I.H. Grant H-5735, and in part by a contract with the U. S. Department of Interior, Fish and Wildlife Service. Presented at the AOCS meeting in Chicago, Ill., 1961.     

Determination of olive oil free fatty acid by fourier transform infrared spectroscopy

A new procedure for determining free fatty acids (FFA) in olive oil based on spectroscopic Fourier transform infrared-attenuated total reflectance spectroscopy measurements is proposed. The range of FFA contents of samples was extended by adding oleic acid to several virgin and pure olive oils, from 0.1 to 2.1%. Calibration models were constructed using partial least-squares regression (PLSR). Two wavenumber ranges (1775–1689 cm⁻¹ and 1480–1050 cm⁻¹) and several pretreatments [first and second derivative; standard normal variate (SNV)] were tested. To obtain good results, splitting of the calibration range into two concentration intervals (0.1 to 0.5% and 0.5 to 2.1%) was needed. The use of SNV as a pretreatment allows one to analyze samples of different origins. The best results were those obtained in the 1775–1689 cm⁻¹ range, using 3 PLSR components. In both concentration ranges, at a confidence interval of α = 0.05, no significant differences between the reference values and the calculated values were observed. Reliability of the calibration vs. stressed oil samples was tested, obtaining satisfactory results. The developed method was rapid, with a total analysis time of 5 min; it is environment-friendly, and it is applicable to samples of different categories (extra virgin, virgin, pure, and pomace oil).     

Simultaneous analysis of low plasma levels of deuterium-labeled saturated and unsaturated fatty acids ast-butyldimethylsilyl esters

A sensitive and accurate method for detection and quantitation of deuterated fatty acids in the presence of large amounts of unlabeled fatty acids is described using mass fragmentography in combination with the preparation of tertiarybutyldimethylsilyl esters (t-BDMS). The method has been applied to determination of deuterated stearic, oleic, elaidic and linoleic acids in human plasma lipoproteins following duodenal perfusion with a micellar mixture of acids. Over a concentration range of 10–1000 ng/ml, the average coefficient of variation for the linoleate was 3% and for the oleate (elaidate) ester was 2%.     

Determination of free fatty acids in palm oil by near-infrared reflectance spectroscopy

A near-infrared (NIR) spectroscopy calibration was developed for the determination of free fatty acids (FFA) in crude palm oil and its fractions based on the NIR reflectance approach. A range of FFA concentrations was prepared by hydrolyzing oil with 0.15% (w/w) lipase in an incubator at 60°C (200 rpm). Sample preparation was performed in Dutch cup, and the spectra were measured in duplicate for each sample. The optimized calibration models were constructed with multiple linear regression analysis based on C=O overtone regions from 1850–2050 nm. The best wavelength combinations were 1882, 2010, and 2040 nm. Multiple correlation coefficients squared (R ²) were: 0.994 for crude palm oil, 0.961 for refined-bleached-deodorized (RBD) palm olein, and 0.971 for RBD palm oil. Calibrations were validated with an independent set of 8–10 samples. R ² of validation were 0.997, 0.943, and 0.945, respectively. The developed method was rapid, with a total analysis time of 5 min, and environmentally friendly, and its accuracy was generally good for raw-material quality control.     

Determination of free fatty acids in crude palm oil and refined-bleached-deodorized palm olein using fourier transform infrared spectroscopy

A rapid direct Fourier transform infrared (FTIR) spectroscopic method using a 100 μ BaF₂ transmission cell was developed for the determination of free fatty acid (FFA) in crude palm oil (CPO) and refined-bleached-deodorized (RBD) palm olein, covering an analytical range of 3.0–6.5% and 0.07–0.6% FFA, respectively. The samples were prepared by hydrolyzing oil with enzyme in an incubator. The optimal calibration models were constructed based on partial least squares (PLS) analysis using the FTIR carboxyl region (C=O) from 1722 to 1690 cm⁻¹. The resulting PLS calibrations were linear over the range tested. The standard errors of calibration (SEC) obtained were 0.08% FFA for CPO with correlation coefficient (R ²) of 0.992 and 0.01% FFA for RBD palm olein with R ² of 0.994. The standard errors of performance (SEP) were 0.04% FFA for CPO with R ² of 0.998 and 0.006% FFA for RBD palm olein with R ² of 0.998, respectively. In terms of reproducibility (r) and accuracy (a), both FTIR and chemical methods showed comparable results. Because of its simpler and more rapid analysis, which is less than 2 min per sample, as well as the minimum use of solvents and labor, FTIR has an advantage over the wet chemical method.