A colorimetric determination of fatty acids as a new assay of lipases in reverse micelles

A new simple spectrophotometric method is described for the determination of the free fatty acid content in triglyceride oils or other lipophilic samples. The method utilizes phenol red as fatty acid indicator, which is solubilized phenol red as fatty acid indicator, which is solubilized in reverse micelles formed by AOT [sodiumbis(2-ethylhexyl) sulfosuccinate] in isooctane. Fatty acid determinations in vegetable oils can be carried out rapidly with oil samples of less than 100 mg. The acid value of three different oils tested agreed quite well with the acid value obtained for the same samples with another colorimetric determination using cupric acetate. The method can be extended to a continuous determination of fatty acids which are released during the initial stage of a lipase catalyzed hydrolysis of triglyceride substrates in reverse micelles. This new sensitive lipase assay has been applied for a lipase ofPseudomonas bacteria with lipase concentrations as low as 0.1 μg/ml. Using trioctanoylglycerol as substrate in 50 mM AOT/isooctane with w_o=[H₂O]/[AOT]=11.1 (pH 9.0), the apparent overall Michaelis-Menten constant (K_m′app,ov) is 27 mM and the turnover number (k_cat) 44 sec⁻¹.     

Free fatty acids in cultured cells

A method for isolation and quantitation of cellular free fatty acid has been developed. When this method was used to quantitate the free fatty acid content of various cells and tissues, their levels of free fatty acids were found to vary over a wide range. In comparing tissue culture cells having different levels of free fatty acid, it was demonstrated that the conditions of culture and the type of serum in the medium are not responsible for the difference in levels. Isotopic studies have shown that the cellular free fatty acid is not biosynthesized, but is derived from the free fatty acid of the medium. Preliminary studies on the fate of the intracellular free fatty acid and a discussion of possible factors controlling the level of this compound in cells are presented.     

Preparation of long-chain fatty acid chlorides

Summary A rapid method has been found for preparing the long-chain fatty acid chlorides, which eliminates purification by distillation. It gave a quantitative yield of product containing less than 1.5% free acid. The method involves treating the free acid with phosphorus pentachloride or trichloride in an inert organic solvent and removing the excess chlorinating agent by washing the solvent phase with water. Phosphorus pentachloride and Skellysolve “F” were preferred for laboratory preparations. For commercial purposes however either chlorinating agent could be used in a variety of inert organic solvents. Infrared analysis was found to give a rapid measure of the free acid content of the prepared acid chlorides. Presented at the fall meeting, American Oil Chemists' Society, Philadelphia, Pa., Oct. 10–12, 1955. Issued as N. R. C. No. 4240.     

Simple, high-efficiency synthesis of fatty acid methyl esters from soapstock

We report a simple method that efficiently esterifies the fatty acids in soapstock, an inexpensive, lipid-rich by-product of edible oil production. The process involves (i) alkaline hydrolysis of all lipid-linked fatty acid ester bonds and (ii) acid-catalyzed esterification of the resulting fatty acid sodium salts. Step (i) completely saponified all glycerides and phosphoglycerides in the soapstock. Following water removal, the resulting free fatty acid sodium salts were rapidly and quantitatively converted to fatty acid methyl esters (FAME) by incubation with methanol and sulfuric acid at 35°C and ambient pressure. Minimum molar reactant ratios for full esterification were fatty acids/methanol/sulfuric acid of 1∶30∶5. The esterification reaction was substantially complete within 10 min and was not inhibited by residual water contents up to ca. 10% in the saponified soapstock. The product FAME contained >99% fatty acid esters, 0% triglycerides, <0.05% diglycerides, <0.1% monoglycerides, and <0.8% free fatty acids. Free fatty acid levels were further reduced by washing with dilute sodium hydroxide. Free and total glycerol were <0.01 and <0.015%, respectively. The water content was <0.04%. These values meet the current specifications for biodiesel, a renewable substitute for petroleum-derived diesel fuel. The identities and proportions of fatty acid esters in the FAME reflected the fatty acid content of soybean lipids. Solids formed during the reaction contained 69.1% ash and 0.8% protein. Their sodium content indicated that sodium sulfate was the prime inorganic component. Carbohydrate was the predominant organic constituent of the solid.     

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.     

Lipase-catalyzed enrichment of long-chain polyunsaturated fatty acids

Lipase hydrolysis was evaluated as a means of selectively enriching long-chain ω3 fatty acids in fish oil. Several lipases were screened for their ability to enrich total ω-3 acids or selectively enrich either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA). The effect of enzyme concentration, degree of hydrolysis, and fatty acid composition of the feed oil was studied. Because the materials that were enriched in long-chain ω3 acids were either partial glycerides or free fatty acids, enzymatic reesterification of these materials to triglycerides by lipase catalysis was also investigated. Hydrolysis of fish oil by eitherCandida rugosa orGeotrichum candidum lipases resulted in an increase in the content of total ω3 acids from about 30% in the feed oil to 45% in the partial glycerides. The lipase fromC. rugosa was effective in selectively enriching either DHA or EPA, resulting in a change of either the DHA/EPA ratio or the EPA/DHA ratio from approximately 1:1 to 5:1. Nonselective reesterification of free fatty acids or partial glycerides that contained ω3 fatty acids could be achieved at high efficiency (approximately 95% triglycerides in the product) by using immobilizedRhizomucor miehei lipase with continuous removal of water.     

Mass determination of the fatty acids released from tannin-stimulated rabbit alveolar macrophages

Previous studies with macrophages that had been prelabeled with [¹⁴C]arachidonic acid (20∶4) have shown that condensed tannin is a potent agonist for the release of arachidonic acid. However, it has not been demonstrated that the percentage release of [¹⁴C]20∶4 accurately reflects the metabolic activity of the endogenous 20∶4 pool. In order to measure the 20∶4 mass release relative to the total cellular 20∶4 pool, the free fatty acids of freshly isolated alveolar macrophages were derivatized with a fluorescent reagent, and then separated and quantified by high-performance liquid chromatography. The amounts of esterified fatty acids were measured by gas chromatography of the methyl esters. Free fatty acid levels were compared to those of the total esterified plus unesterified fatty acids to determine the actual percentage released of each fatty acid. Tannin-stimulated release of 20∶4 mass reflected that previously reported for the release of [¹⁴C]20∶4 label but at a slower rate and at a much lower percentage indicating that [¹⁴C]20∶4 had been incorporated into part of a more reactive pool. The specificity of the fatty acid release induced by tannin and β-1,3-glucan, a known agonist for 20∶4 release, was also examined. Both agonists promoted an increase in the levels of free 20∶4 and of other fatty acids. A comparison of the absolute increases of each of the fatty acids indicated that tannin caused a preferential increase in the mass of free 20∶4, whereas β-1,3-glucan evoked a selective increase in the mass of 16∶0. Deceased.     

Some aspects of fatty acid metabolism in brown adipose tissue

The action of catecholamines on white and brown adipose tissue is compared. The available evidence indicates that lipolysis is initiated in both tissues by hormonal action. There are, however, some differences in the behavior of the lipolytic process in the two tissues in their response to theophylline, nicotinic acid and insulin which remain unexplained. It would appear that the release of free fatty acids triggers the stimulation of O₂ consumption in both tissues. In brown adipose tissue no evidence has been obtained to indicate that the increased O₂ consumption is geared to the production of ATP for the purpose of reesterification of the released free fatty acids, as is the case in white adipose tissue. One of nine papers to be published from the Symposium “Brown Adipose Tissue,” presented at the AOCS-AACC Joint Meeting, Washington, D.C., March 1968.     

Separation of fatty acids/triacylglycerol by membranes

Separation of fatty acids from triacylglycerol by membrane separation technique has been studied. Mixtures of triacylglycerols and fatty acids were extracted with alcohol, and the alcohol extracts were treated for recovery of oil by membrane separation technique. The membranes used were of both cellulosic and noncellulosic types. Polyamide membranes showed better selectivity toward fatty acid separation as compared to cellulose acetate and polysulfone membranes. For groundnut oil/fatty acid/alcohol mixture and a polyamide membrane, the free fatty acid (FFA) concentration in the permeate was 86.82% at 0.7 MPa pressure when the feed had 61.71% FFA. A reasonable permeate flux of 67.36 Im⁻²h⁻¹ was obtained. Results obtained have been useful in selecting membrane material suitable for such applications.     

Chemoenzymatic epoxidation of unsaturated fatty acid esters and plant oils

In the presence of an immobilized lipase fromCandida antacrtica (Novozym 435^R) fatty acids are converted to peroxy acids by the reaction with hydrogen peroxide. In a similar reaction, fatty acid esters are perhydrolyzed to peroxy acids. Unsaturated fatty acid esters subsequently epoxidize themselves, and in this way epoxidized plant oils can be prepared with good yields (rapeseed oil 91%, sunflower oil 88%, linseed oil 80%). The hydrolysis of the plant oil to mono- and diglycerides can be suppressed by the addition of a small amount of free fatty acids. Rapeseed oil methyl ester can also be epoxidized; the conversion of C=C-bonds is 95%, and the composition of the epoxy fatty acid methyl esters corresponds to the composition of the unsaturated methyl esters in the substrate. Based partly on a lecture at the 86th AOCS Annual Meeting & Expo, San Antonio, Texas, May 7–11, 1995.     

The influence of storage on the fatty acid content of cod liver oil

Summary A study has been made of the change of fatty acid content of cod liver oils during storage. Thirty oils varying in acid content from 0.38% to 20.88% were procured in the open market. They represented three types of oils—medicinal, animal feeding and industrial oils. The samples of oil were stored at room temperature, in the dark, in three-fourths filled, four-ounce, amber, cork-stoppered bottles. The acid content was determined twelve times during a period of forty-two months. The acid content of each oil increased. The amount of increase varied from 7.42% for a sample of industrial oil with an initial acid content of 16.45% to 84.80% for a sample of animal feeding oil with an initial acid content of 0.99%. In general, the cod liver oils with initial high acid content showed a lower percentage increase in free fatty acids than oils with a low initial free fatty acid content.     

超声微波耦合催化合成蔗糖脂肪酸酯

通过超声微波耦合作用,采用无溶剂法制备蔗糖脂肪酸酯,研究催化剂类型、催化剂用量、超声微波功率、酯糖物质的量比和反应温度对蔗糖脂肪酸酯产率的影响。结果表明,超声微波耦合对蔗糖与脂肪酸甲酯的酯交换反应具有良好的促进作用,超声微波耦合作用下,蔗糖和脂肪酸甲酯能够形成较为良好的乳化状态。有机钛催化剂对蔗糖与脂肪酸甲酯的酯交换反应具有极佳的催化性能,与碱催化剂相比,具有用量少和活性高的特点。以钛酸四异丙酯为催化剂,在超声微波耦合体系中,蔗糖脂肪酸酯最高产率可达86.6%,其中,产品单酯含量为92.7%。     

Origin of the high saturated fatty acid content of rat fecal lipids

The biohydrogenation of unsaturated fatty acids and the preferential absorption of unsaturated fatty acids over long chain saturated fatty acids from the gut have been investigated to find the origin of the high saturated fatty acid content of the facal lipids of rats fed soybean oil. Label from dietary (1-¹⁴C)-linoleic acid was recovered in the saturated and monounsaturated fatty acids of the fecal lipid. However, when (9,10-³H)-stearic acid and (1-¹⁴C)-linoleic acid were fed together, the isotope ratio (³H/¹⁴C) of the fecal lipid was 1.9 times that of the diet. It is concluded that both processes occur.     

Quantitative analysis of free and bound fatty aldehydes: Optimum conditions for p-nitrophenylhydrazone formation

A method is described for the analysis of free and bound fatty aldehydes in lipid extracts. By radiolabeling techniques it was shown that the method for measuring free fatty aldehydes when used in the presence of bound fatty aldehydes measures less than 1% of the bound aldehydes. Parameters of time, temperature and optimum acid concentration are reported. A comparison has been made between the present method and other published methods of measuring p-nitrophenylhydrazones with emphasis on recoveries and reactivities. The present method is suitable for measuring as little as 0.02 μmoles of fatty aldehyde. The method has been applied to the analysis of free and bound aldehydes present in various mouse tissues.     

Further studies on the pancreatic hydrolysis of some natural fats

A series of animal and vegetable fats has been subjected to hydrolysis with pancreatic lipase. From the results obtained, the triglyceride compositions of the original fats have been calculated by the method previously proposed by Coleman and Fulton. These compositions show substantial agreement with those obtained by other methods. Similarities and differences between fats are shown to be reflections of similarities and differences in glyceride composition. The middle position of the triglycerides has been shown to be largely occupied by unsaturated acids in the case of shea, illipé, and cocoa butters; and by palmitic acid in the case of lard. From a study of the glyceride compositions of a series of lards, of differing fatty acid content, it is suggested that the glyceride composition of a lard may be predicted from a knowledge of its fatty acid content, alone. It is concluded that the hydrolysis procedure, and the method of interpreting the results proposed, together provide a simple and rapid method for the analysis of natural fats. *** DIRECT SUPPORT *** A03O2180 00002     

Effect of free carboxylic groups on the course of sulfur trioxide sulfonation of unsaturated fatty acids

Summary The direct action of one mole of sulfur trioxide in liquid sulfur dioxide at atmospheric pressure at −10 to −9°C. on one mole of unsaturated fatty acids such as oleic acid, undecylenic acid, and crotonic acid yielded 85–90% of sulfonated products in which a monosulfonated derivative, rich in residual carbon to carbon unsaturation, predominates. Hydroxy sulfonates and sulfonated-sulfated products, postulated to form from carbyl sulfate intermediates, are present in lesser amounts. Esters of unsaturated fatty acids, such as n-propyl oleate, require at least two moles of sulfur trioxide for 80–90% yields of sulfonated product. These sulfonated products are postulated to form almost exclusively through the carbyl sulfate intermediate. The free carboxyl group apparently determines the course of sulfonation. Its action is believed to be that of first forming an acyl sulfate or mixed anhydride with sulfur trioxide, followed by hydrogen replacement in a methylene group, probably in an allyl position to the double bond, with a sulfonic acid group. The mechanism and nature of products resemble those previously observed by Suter in the dioxane-sulfur trioxide sulfonation of methallyl chloride (6). The sulfonated unsaturated reaction products from oleic and undecylenic acids exhibit lower double bond reactivity toward hydrogen and halogen addition than the unsulfonated starting materials. The monosulfonated oleic acid, because of its stability toward hydrolysis, holds promise as an important surface-active agent, particularly under acidic conditions. Patent covering the reaction products and their preparation has been applied for.     

Rapid and direct quantitative analysis of positional fatty acids in triacylglycerols using 13C NMR

There is increasing evidence that the positional fatty acid composition (FAC) of TAG is more important than total FAC with regard to nutrition values of edible oils and fats. A rapid and direct regiospecific analysis of positional fatty acids in TAG using ¹³C NMR was developed to overcome the tedious conventional methods which involve enzymatic hydrolysis, Grignard chemical degradation, and chromatography analysis. A set of NMR data acquisition parameters and processing methods had proven their excellent versatility and applicability on various types of oils and fats, with systematic error of 1.0 mol%. It was found that there are discrepancies between the regiospecific analysis results obtained by the current method and by conventional methods. Probable acyl migration occurring in the hydrolysis process in conventional methods is a noted problem. As the current ¹³C NMR method is a direct measurement and no hydrolysis of the sample is needed, acyl migration during the analysis is eliminated. As a result, the saturation level is always higher at sn‐1, 3 positions and lower at sn‐2 position in TAG structure of oils in the regiospecific data obtained from the current ¹³C NMR than that from conventional methods. In the absence of laborious chemical derivatization, this method is simple, accurate, and user‐friendly for researchers, especially for nutritionists to support their nutritional studies from the perspective of positional FAC of edible oils and fats.     

Modification of Vegetable oils. IV. Reesterification of fatty acids with glycerol

Summary 1. An investigation has been made of the esterification of glycerol and peanut oil fatty acids under reduced pressure, with and without the assistance of various metal chlorides and oxides as catalysts. 2. The uncatalyzed reaction is bimolecular in character but proceeds in two successive stages, of which the latter has the lower velocity constant. Velocity constants have been determined for the initial and final stages of the reaction, at intervals between 166° and 241° C. The calculated heats of activation for the initial and final stages of the reaction are respectively 12,300 and 10,800 calories per mole. The free fatty acid concentration corresponding to the termination of the first stage decreases progressively as the temperature of the reaction is increased. 3. Of a wide variety of metal oxides and chlorides tested, zinc and tin chlorides were outstanding in catalytic activity. The reaction, when catalyzed with these materials, is complex and no longer simply bimolecular. It is believed that tin and zinc chlorides react initially with free fatty acids and free glycerol to form metal soaps and chlorohydrins, and that esterification proceeds through interaction of these two initial reaction products. Other metal chlorides, including the chlorides of aluminum, antimony, mercury, nickel, magnesium, manganese, lead, iron, and cadmium, do not appear to be capable of reacting in this manner, and are relatively poor catalysts. The oxides of tin and zinc are also deficient in catalytic activity, as is hydrochloric acid. 4. The reaction proceeds at a reasonable speed, i.e., the FFA content of the product is reduced to about 3% in 6 hours, if 0.0008 mole of tin chloride per 100 g. of fatty acids is used as a catalyst at 175° C. or if a similar amount of zinc chloride is used as a catalyst at 200° C. Equally rapid esterification is obtained without a catalyst only above 250° C. Esterification is assisted by maintaining a vacuum upon the reaction vessel to remove water vapor from the reacting material as rapidly as it is formed. A vacuum of about 20 mm. pressure of mercury is satisfactory. 5. If zinc or tin chloride catalysts are employed, the metals may be completely removed from the esterified oils by ordinary alkali refining. These catalysts do not cause the oil to polymerize during the course of esterification, do not cause conjugation in the oils, and are not detrimental to the color of the product. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Germination and free fatty acids in seed stock lots of cottonseed

Summary The relationship of the free fatty acid content in the oil to the percentage germination for 254 samples of cottonseed of different varieties indicates that the free fatty acid content may be used as a practical screening index for use in selecting lots of cottonseed to be reserved for seeding purposes and subsequent testing for germination. The percentage germination decreases in general with increasing free fatty acid content. The mathematical probability that a given lot of seed will exceed a specified minimum germination value decreases markedly as the free fatty acid content of the oil increases. Insofar as practical, it is suggested that cottonseed reserved for seeding have a low free fatty acid content, less than 0.75% in the oil if at all possible. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture. In cooperation with the Production and Marketing Administration, U. S. Department of Agriculture, under Marketing Act of 1946.     

Antagonism of [3H]fatty acid incorporation into vimentin by sodium pyruvate: Pitfalls of protein acylation

In the course of studying possible fatty acid acylation of vimentin by cultured bovine lens epithelial cells, several potential pitfalls of protein-fatty acid acylation were recognized. Even exhaustive delipidation of vimentin with organic solvents failed to remove all noncovalently associated [³H]palmitate and [³H]myristate. Hydroxylamine treatment of vimentin, separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), failed to remove either palmitate or myristate derived radiolabel. Hydroxylamine treatment did remove palmitate label from a group of lower molecular weight proteins. The myristate radiolabel associated with vimentin recovered after SDS-PAGE and subjected to acid hydrolysis was shown due to incorporated [³H]amino acids, mainly glutamic acid, generated from the fatty acid. Adding excess sodium pyruvate to labeling media has been used by others to reduce the metabolic conversion of fatty acids to amino acids; however, no direct evidence in support of this antagonism was presented. We observed that inclusion of sodium pyruvate at between 5 and 20 mM in the labeling medium produced a dramatic decrease in incorporation of myristic acid radiolabel into vimentin. However, inclusion of even 20 mM pyruvate did not completely antagonize the metabolic conversion of fatty acid label to amino acids. Furthermore, the sodium pyruvate antagonism could be totally obscured if the exposure of X-ray film by fluorography was even slightly prolonged. The results illustrate the danger in assuming that solvent extraction totally delipidates proteins and that adding sodium pyruvate to labeling media prevents the transfer of fatty acid label to amino acids. Caution is necessary to conclude that radiolabel associated with specific proteins following incubation of cells with labeled fatty acid is due to covalent attachment of the fatty acid to the protein.