Final Report for FW08-033
A simplified protocol was developed to obtain fatty acid methyl esters (FAME) from any sample. The method consists of two steps, conducted in a single reaction tube. The protocol relies on the presence of water, which heretofore was eliminated in FAME synthesis methods.
A simplified protocol to obtain FAME directly from fresh tissue, oils, or feedstuffs, without prior organic solvent extraction was presented. With this protocol, FAME synthesis is conducted in the presence of up to 33% water. Wet tissues, or other samples, are permeabilized and hydrolyzed for 1.5 hr at 55C in 1N KOH in methanol containing C13:0 as internal standard. The KOH is neutralized and the free fatty acids are methylated by H2SO4 catalysis for 1.5 hr at 55C. Hexane is 10 added to the reaction tube, which is vortex-mixed and centrifuged. The hexane is pipette into a GC vial for subsequent gas chromatography. All reactions are conducted in a single screw cap Pyrex tube for convenience. The methods meets a number of criteria for fatty acid analysis including not isomerizing conjugated linoleic acid (CLA) or introducing fatty acid artifacts. It is applicable to fresh, frozen, or lyopholyzed tissue samples, in addition to oils, waxes, and feedstuffs. The method saves time and effort and is economical when compared to other methods. Its unique performance, including easy sample preparation, is achieved because water is included in the FAME reaction mixtures rather than eliminated.
The analysis of fatty acids has become increasingly important because more people have become aware of their nutritional and health implications. Because of this, a method for analyzing fatty acids that provides both rapid and reliable results is of great value. Many methods are currently used to analyze fatty acids. These methods are not necessarily convenient, nor direct, and often must be optimized for reaction conditions. On the other hand, the ideal method would determine the total fatty acid concentration in tissues, oils, and feed samples by converting fatty acid salts, as well as the acyl components in all lipid classes such as triacylglycerols, phospholipids, sphingolipids, and waxes, to methyl esters using a simple direct one-step esterfication procedure.
In this paper we present a method that is established upon a surprising conception: We added water to the fatty acid methyl ester (FAME) synthesis reagents. Until now, FAME synthesis methods have rigorously avoided water as a matter of standard procedure. However, by adding water the dynamics of sample preparation and methyl ester formation can be revisited and the ideal outcome of FAME synthesis becomes possible. Although the method described herein requires two steps, it does so in one reaction tube.
The presence of water in the reagents allows for tissue sample dissolution and facilitates the total extraction of fatty acids not possible in the absence of water. Water does not interfere with the methylation of any fatty acid. The method uses familiar and economical chemicals, namely, methanol, potassium hydroxide, and sulfuric acid, but uses these methylating reagents in the presence of water. As a result, of these characteristics, the direct FAME synthesis method is convenient, reliable, and efficient.
Our primary objective is to develop a method to directly methylate fatty acids from muscle tissue, oils, and feedstuffs in aqueous solution.
MATERIALS AND METHODS
To assess certain features of our method, we compared direct FAME synthesis to two methylating agents routinely used for FAME synthesis, namely the base catalyst sodium methoxide and the acid catalyst boron trifluoride.
The samples we used in this manuscript were chosen for distinct reasons. The Supelco fatty acid standard mixture was chosen because it contained short and long chain saturated, monounsaturated, and polyunsaturated fatty acids in defined amounts and thus served as a primary test of the feasibility of our method. Fish oil was chosen because it is an important source of the long chain plyunsaturated omega-3 esterfied eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic (DHA) fatty acids. Conjugated linoleic acid (CLA), as the free acid, was chosen because it is of medical importance as perhaps the only fatty acid that can directly inhibit cancer in animal models and because current FAME synthesis methods often cause undesirable isomerizations of this fatty acid. Beef longissimus muscle was chosen because of our special interest in beef fatty acids, and it serves as direct test of the ability of direct FAME synthesis to extract and methylate all of the fatty acids present in meat tissue.
Direct FAME synthesis is a two-step procedure. In the first step, sample fatty acid esters are hydrolyzed to free fatty acids, and in the second step the free fatty acids are converted to FAME. When the first step of direct FAME synthesis was applied to the Supelco standard FAME mixture, the esters were hydrolyzed to free fatty acids that were not volatile enough to enter the GC column. These results, the absence of fatty acid peaks, provided formal evidence that the first step in direct FAME synthesis completely hydrolyzed the Supelco standard FAME to free fatty acids, which was the desired general prerequisite for the subsequent methylation step of direct FAME synthesis.
Then the second step of direct FAME synthesis, the methylation step, was applied to the Supelco free fatty acids produced by the first step. All of the GC peaks present in the original Supelco standard mixture were again observed, as can be seen by comparing the fatty acids of direct FAME synthesis to those of the Supelco mix. When presented with a FAME sample, as in this experiment, both sodium methoxide and boron trifluoride likewise gave the same FAME values present in the original Supelco mix.
Fatty acid artifacts are a concern in fatty acid analysis. Since direct FAME synthesis, sodium methoxide, or boron trifluoride did not generate new fatty acid peaks, no fatty acid artifacts were created in the Supelco standard FAME mixture.
Care should be taken in the evaporation of solvents as appreciable amounts of esters up to C14 can be lost if this step is performed carelessly. The vigorous use of nitrogen to evaporate solvents must be avoided. Boron trifluoride in methanol has a limited shelf life, even when refrigerated, and the use of old or too concentrated solutions often resulted in the production of artifacts and the loss of appreciable amounts of polyunsaturated fatty acids by addition of methanol across the double bonds.
All of our results show the efficacy of the direct FAME synthesis method, which allows up to 33% water content.
Direct FAME synthesis is convenient. Since water is part of the method, and not antagonistic to it, sample preparation is rapid; one only weighs-out or pipets the sample into a Pyrex tube and then conducts the direct FAME synthesis. Gone is the preparation time it takes to lyophilize a sample (usually days), or the prior organic solvent extractions and nitrogen evaporations (usually hours) that are required to eliminate water in the other fatty acid methods.