An enzymic cycling method for the determination of free fatty acids with acyl-CoA synthetase and acyl-CoA hydrolase

A method for measuring free fatty acids by enzymic cycling is described. Free fatty acids are converted to acyl-CoAs by acyl-CoA synthetase, then the acyl-CoAs are hydrolyzed back to the free fatty acids by acyl-CoA hydrolase in a cyclic fashion. The amounts of AMP produced during this cyclic reaction are determined from the absorbance at 340 nm in the presence of AMP deaminase and glutamate dehydrogenase. This method is sensitive to as low as 0.1 nmol of free fatty acids, and the standard curve is linear up to 1.0 nmol. This method shows a broad specificity for long-chain fatty acids (C12--C20) and the recoveries of fatty acids added to bacterial cell-free extracts are more than 90%.     

A modified assay system for enzymes involved in N-acetyl group transfer reactions: its use to study enzymes involved in ornithine biosynthesis in plants

An enzymatic method for the determination of the amount of free fatty acids released from triglyceride by lipoprotein lipase is described. The quantity of free fatty acids present in media before and after incubation is measured spectrophotometrically by the oxidation of NADH in the final reaction of a series of coupled enzymatic reactions. This assay for lipoprotein lipase is unlike previously described assays in that radioactive substrates or titration procedures are not used in the free fatty acid determination. In addition, another method for assay of lipoprotein lipase activity that involves the separation of free fatty acids from triglycerides by adsorption chromatography with Florisil as a stationary phase is described.     

Free fatty acid determination by peroxidase catalysed luminol chemiluminescence

A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl-CoA oxidase. The H₂O₂ formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C₁₀-C₁₈) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H₂O₂ standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase-luminol reaction.     

Validation of a new procedure to determine plasma fatty acid concentration and isotopic enrichment.

Assessment of free fatty acid (FFA) concentration and isotopic enrichment is useful for studies of FFA kinetics in vivo. A new procedure to recover the major FFA from plasma for concentration and isotopic enrichment measurements is described and validated. The procedure involves extraction of plasma lipids with hexane, methylation with iodomethane (CH(3)I) to form fatty acid methyl esters (FAME), and subsequent purification of FAME by solid phase extraction (SPE) chromatography. The new method was compared with a traditional method using thin-layer chromatography (TLC) to recover plasma FFA, with subsequent methylation by BF(3)/methanol. The TLC method was found to be less reliable than the new CH(3)I method because of contamination with extraneous fatty acids, chemical fractionation of FFA species, and incomplete recovery of FFA associated with TLC. In contrast, the CH(3)I/SPE method was free of contamination, did not exhibit chemical fractionation, and had higher recovery. The iodomethane reaction was specific for free fatty acids; no FAME were formed when esterified fatty acids (triglycerides, cholesteryl esters, phospholipids) were subjected to the methylation reaction.We conclude that the CH(3)I/SPE method provides rapid and convenient recovery of plasma fatty acids for quantification or GC/MS analysis as methyl esters, and is not subject to the problems of contamination, reduced recovery, and chemical fractionation associated with recovery of FFA by TLC.     

Enzymatic determination of serum-free fatty acids: a colorimetric method

A simple and sensitive enzymatic method is described for the determination of serumfree fatty acids. The method is based on the activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3). The reaction is followed as the production of hydrogen peroxide by using acyl-CoA oxidase/peroxidase/4-aminoantipyrine/phenol system. Results on human sera correlate well with those obtained by our previously described enzymatic method (Shimizu et al. (1979) Anal. Biochem.98, 341–345) and the chemical colorimetric method.     

Bioluminescent determination of free fatty acids

A simple, highly specific, and sensitive bioluminescent method for determination of free fatty acids in unextracted plasma or serum has been developed. The method is based on the activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3). The pyrophosphate formed is used to phosphorylate fructose 6-phosphate in a reaction catalyzed by the enzyme pyrophosphate-fructose-6-phosphate phosphotransferase (EC 4.1.2.13). The triosephosphates produced from fructose 1,6-bisphosphate by aldolase are oxidized by NAD in the presence of arsenate to 3-phosphoglycerate. The NADH is detected via the bacterial NADH-linked luciferase system. Excellent agreement has been obtained by comparison with accepted methods. In addition, for the determination of serum free fatty acids, the method is particularly applicable for following lipolysis of isolated adipocytes.     

Separation and preparative purification of arachidonic acid from microbial lipids by urea inclusion reaction and HPLC

Arachidonic acid (AA) was separated and purified from microbial lipids by the combined method of urea inclusion reaction and reversed-phase high performance liquid chromatography. At first, AA was concentrated from free fatty acids made from microbial lipids by a urea inclusion reaction. The optimum conditions were as follows: methanol was the suitable solvent, the ratio of free fatty acids to urea to methanol was 1:2:8 (wt/wt), and the temperature of the urea inclusion reaction was -10 degrees C. The AA content was increased from 38% to 79%, and then AA was purified on a C(18) preparative column (300 mm x 30 mm I.D., d(p)=15 microm), using methanol-water (95:5, v/v) as the mobile phase, at a flow rate of 5 mL/min. The purity of AA after two steps purification reached 99%. This result indicates that the combined method of the urea inclusion reaction and reversed-phase high performance liquid chromatography is a promising technique for purification of AA.     

Microwave-mediated analysis for sugar, fatty acid, and sphingoid compositions of glycosphingolipids

For chemical characterization of glycosphingolipids, it is necessary to determine the chemical compositions of three constituents, i.e., sugars, fatty acids, and sphingoids. A new rapid analytical method is described using a one-pot reaction in a household microwave oven, producing sugars, fatty acids, and especially sphingoids free of by-products, from a single aliquot of a biological sample. Glycosphingolipids were hydrolyzed by microwave exposure with 0.1 M NaOH/CH(3)OH for 2 min followed by 1 M HCl/CH(3)OH for 45 s. The alkaline methanolysis step produced intermediate lysoglycosphingolipids virtually free of by-products such as the O-methyl ethers usually seen. The fatty acid methyl esters were extracted with n-hexane, and other reaction products were dried, taken up in aqueous alkaline methanol, and shaken with chloroform. Sphingoids partitioned into the organic phase under these conditions, whereas the sugar portion that partitioned into the aqueous phase was re-N-acetylated and remethanolyzed for 30 s by microwave exposure. Analysis of the profiles of glycosphingolipid constituents obtained using the microwave oven method showed that they were quantitatively and qualitatively comparable to those obtained by time-consuming conventional methods, which require reaction for several hours. Analysis of the three constituents, including analysis by gas chromatography, may be obtained within 1 day using the method described here.     

A method for specific analysis of free fatty acids in biological samples by capillary gas chromatography.

The present report describes a simple method to selectively extract free fatty acids and analyze them by capillary gas-liquid chromatography. The procedure is based on the use of fumed silicon dioxide. In the presence of plasma, this material induces a rapid rise in the viscosity of the mixture and presents the ability to trap large particles such as emulsified lipids and lipoproteins. Albumin-bound fatty acids are thus left in the aqueous media. We present applications of our procedure for the analysis of free fatty acids in 0.2 ml of plasma from rat or human. By comparison with the method utilizing thin-layer chromatography for the separation of fatty acids and gas chromatography analysis, the present method has been found to be reliable and simple. The recovery of linoleic acid was 92.1 +/- 8.2%, a value which is about twice better than that obtained with the procedure using thin-layer chromatography. In particular, long-chain polyunsaturated fatty acids were better preserved. Our procedure does not require the use of organic solvents and its simplicity and reproducibility make it suitable for routine specific determination of the composition of free fatty acids in biological samples.     

Quantitative analysis of free fatty acids in rat plasma using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with meso-tetrakis porphyrin as matrix

Quantitative analysis of free fatty acids was achieved using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a meso-tetrakis porphyrin matrix. Cesium acetate was employed as a cationizing agent. The MALDI signal was reproducible and dominated by cesiated cesium carboxylates [RCOOCs + Cs]+. The addition of two Cs ions resulted in a mass shift of 264.8 Da for each fatty acid and greatly reduced background peaks. A linear relationship between fatty acid concentration and corresponding fatty acid to internal standard peak intensity ratio was observed for three representative fatty acids analyzed across a concentration range from 4.40 to 150 microM, with correlation coefficients between 0.986 and 0.987. The application of this method was demonstrated with the analysis of free fatty acids in nonfasted and fasted rat plasmas. A total of eight free fatty acids (14:0, 16:0, 16:1, 17:0, 18:0, 18:1, 18:2, and 20:4) were detected. The relative peak height ratios of the fatty acids to the internal standard allow quantitative measurements of the free fatty acids. It was shown that the levels of free fatty acids were higher in fasted rats than in rats in a nonfasted state. This method is simple, sensitive, and fast. Thus, it provides an appealing tool for the analysis of free fatty acids or other low-molecular weight compounds during drug discovery and/or development.     

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.     

Effects of free fatty acids on fibrinolytic activity.

A novel method for the estimation of fibrinolytic activity is proposed. In this method, a fibrin clot suspension is used as a substrate (fibrin is known to be a physiological substrate of plasmin). The fibrin clot suspension was prepared by homogenization of human fibrin clots. With this method, we found that free fatty acids inhibited the plasmin activity, and long-chain, unsaturated free fatty acids had a particularly strong inhibitory action on plasmin. As regards the mechanism of the inhibitory action, free fatty acids may not inhibit complex formation between plasmin and fibirin, but may make it impossible for plasmin to act on fibrin due to deformation of the surface of the fibrin clot.     

LIBERATION OF 5,8,11,14,17-EICOSAPENTAENOIC ACID AND OTHER POLYUNSATURATED FATTY ACIDS FROM LIPIDS AS A GRAZER DEFENSE REACTION IN EPILITHIC DIATOM BIOFILMS

Acute grazer toxicity of freshwater diatom biofilms was determined using Thamnocephalus platyurus Packard, an anostracan grazer, as the bioassay organism. The diatoms exhibited toxicity when the cells of the biofilm were freeze–thawed before the assay. The toxicity could be extracted from the biofilms with methanol and acetone, and only minimal toxicity was left in the insoluble residue. Bioassay-guided HPLC separation of the methanolic extract was performed to trace the most toxic components. Analysis by UV spectrometry, gas chromatography, and mass spectrometry showed that 5,8,11,14,17-eicosapentaenoic acid was responsible for most grazer toxicity. The 24-h LC₅₀ of this polyunsaturated fatty acid was 34 μM in the Thamnocephalus platyurus bioassay. The concentrations of other free fatty acids were not high enough to contribute significantly to the toxicity. Procedures that affected the integrity of the cells (e.g. solvent extraction, freezing and thawing, osmotic stress by addition of 20% NaCl, or grinding the cells in a mortar) were taken as model reactions for grazing and had the common effect of resulting in a dramatic increase of free polyunsaturated and saturated fatty acids. Under these conditions, about 30% of the total fatty acids of the diatoms was transformed from the bound into the free form. The time necessary for liberation was very short. With the exception of 5,8,11,14,17-eicosapentaenoic acid, which continued to be liberated, the hydrolysis of the other fatty acids was terminated less than 1 min after initiating the reaction. The classical extraction procedures using methanol and other solvents led to the appearance of a high percentage of free fatty acids in live cells. Treatment of biofilms with these solvents did not stop the hydrolysis of lipids initiated by the disintegration of the cells. However, boiling acetone completely suppressed the hydrolytic reactions, and free polyunsaturated fatty acids were not detected in live biofilm organisms, although nontoxic saturated fatty acids were present in moderate concentrations. These results were interpreted as an indication that the frequently reported existence of free polyunsaturated fatty acids in live biomass is an analytical artifact.     

Utilization of porcine pancreatic phospholipase A2 for the preparation of a marine fish oil enriched in (n - 3) polyunsaturated fatty acids

A simple and rapid method is described for the preparation of a marine oil fraction highly enriched in (n - 3) polyunsaturated fatty acids. Cod roe, containing lipid up to 15% of its dry weight, approximately 70% of which is phospholipid, was the starting material. Incubation of a concentrated aqueous extract of the roe with porcine pancreatic phospholipase A2 (EC 3.1.1.4) was the key step in the procedure. Extraction of the freeze-dried reaction product with diethyl ether containing formic acid produced an oil in a yield of 1.0 g/100 g wet wt of starting roe. The oil contained over 95% as free fatty acids, with 20:5 (n - 3) and 22:6 (n - 3) accounting for up to 24 and 40%, respectively, of the total free fatty acids. The therapeutic use of the oil is mentioned.     

Automated modification of Duncombe's method for the ultramicro determination of serum free fatty acids.

An automated continuous-flow method is described for estimating free fatty acids in serum using 25-mul samples. The procedure depends on the formation of copper soaps on the surface of a semipermeable membrane, their transfer into chloroform, and subsequent determination of dissolved copper. The membrane separating copper reagent and chloroform is supported between thin-channel dialysis plates. Chloroform extracts of serum free fatty acids are passed through the dialysis unit, and dissolved copper in the outflow is estimated colorimetrically. The procedure gives values that agree with a standard titrimetric method.     

A reliable analysis of tissue free fatty acids by gas-liquid chromatography

A convenient and reliable gas-liquid chromatographic method for determining the free fatty acids in biological specimens is described. The free fatty acids were extracted with hexane in the presence of H3PO4 and then back-extracted from the hexane phase into a very small volume of trimethyl (alpha, alpha, alpha-trifluoro-m-tolyl)ammonium hydroxide solution. Direct injection of the resultant quaternary ammonium salts of the fatty acids into a gas-liquid chromatograph unit gave their methyl esters, with a high recovery. The presence of triglycerides, phospholipids, or cholesterol esters did not interfere with the determination of free fatty acids. This method was applied to determination of free fatty acids in the samples of serum or brain. The results were more precise and reliable than those reported with the conventional methods with TLC separation. This method should be a useful aid for providing precise information about the physiological or pathological roles of free fatty acids.     

Lipoxygenase-dependent degradation of storage lipids

Oilseed germination is characterized by the mobilization of storage lipids as a carbon source for the germinating seedling. In spite of the importance of lipid mobilization, its mechanism is only partially understood. Recent data suggest that a novel degradation mechanism is initiated by a 13-lipoxygenase during germination, using esterified fatty acids specifically as substrates. This 13-lipoxygenase reaction leads to a transient accumulation of ester lipid hydroperoxides in the storage lipids, and the corresponding oxygenated fatty acid moieties are preferentially removed by specific lipases. The free hydroperoxy fatty acids are subsequently reduced to their hydroxy derivatives, which might in turn undergo beta-oxidation.     

Investigations on the Influence of a Low Casein Diet on the Free Fatty Acids in Liver Homogenate by a Method with Some Devices in Purification Procedure Using KOH

Previously, some changes were noticed in energy metabolism of rats fed a low casein diet. In connection with these phenomena, influence of a low casein diet on the composition and amounts of free fatty acids in liver homogenate after autolyzing for a few hours was investigated. For the measurement of free fatty acids, they were purified by a method with some devices in purification procedure using KOH. It was found that amounts of free fatty acids in liver homogenate after autolyzing for a few hours were lower in rats fed a low casein diet.     

Enzymatic lipid removal from surfaces--lipid desorption by a pH-induced "electrostatic explosion"

Removal of lipidic molecules from surfaces can be accomplished using detergents containing lipases. Surface cleaning is usually performed under alkaline conditions due to increased solubility of the hydrolysis products, especially free fatty acids. This paper shows that removal of a triacylglycerol film from a surface can be dramatically enhanced in a sequential system where pH is shifted to alkaline conditions after an initial lipolytic reaction period at or below neutral pH. Data from three different biophysical techniques, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), quartz crystal microbalance with dissipation monitoring (QCM-D), and total internal reflection fluorescence spectroscopy (TIRF) clearly show the effects of such cleaning procedure. Initially the reaction is carried out at pH below the pKa value of the fatty acids formed upon triacylglycerol hydrolysis, and the protonated fatty acids accumulate in the film. The mechanism of lipid removal, induced by increasing pH to a value above the fatty acid pKa, is explained by a burst caused by electrostatic repulsion between rapidly ionised fatty acids, i.e. by an "electrostatic explosion". Performing the initial hydrolysis at pH 6 and the subsequent rinse at pH 10, using triolein as model substrate, lipid removal from surfaces by both commercial detergent lipases and non-commercial lipases was significantly improved compared to a reaction at constant pH 10.     

Microsomal preparations from plant and yeast acylate free fatty acids without prior activation to acyl-thioesters

Acylation of fatty acids to hydroxy groups in cells generally require activation to a thioester (ACP or CoA) or transacylation from another oxygen ester. We now show that microsomal membranes from Arabidopsis leaves efficiently acylate free fatty acids to long chain alcohols with no activation of the fatty acids to thioesters prior to acylation. Studies of the fatty alcohol and fatty acids specificities of the reaction in membranes from Arabidopsis leaves revealed that long chain (C18-C24) unsaturated fatty alcohols and C18-C22 unsaturated fatty acids were preferred. Microsomal preparations from Arabidopsis roots and leaves and from yeast efficiently synthesized ethyl esters from ethanol and free fatty acids. This reaction also occurred without prior activation of the fatty acid to a thioester. The results presented strongly suggest that wax ester and ethyl ester formation are carried out by separate enzymes. The physiological significance of the reactions in plants is discussed in connection to suberin and cutin synthesis. The results also have implication regarding the interpretation of lipid metabolic experiments done with microsomal fraction.