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J. V. O'Fallon, J. R. Busboom, M. L. Nelson and C. T. Gaskinstissues, oils and feedstuffs
A direct method for fatty acid methyl ester (FAME) synthesis: Application to wet meat
published online February 12, 2007J ANIM SCI
http://jas.fass.org/content/early/2007/02/12/jas.2006-491.citationthe World Wide Web at:
The online version of this article, along with updated information and services, is located on
www.asas.org
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Direct Fame synthesis 1
Running head: Direct FAME synthesis 1
2
3
A direct method for fatty acid methyl ester (FAME) synthesis: Application to wet 4
meat tissues, oils and feedstuffs 5
6
7
J. V. O’Fallon, J. R. Busboom, M. L. Nelson, and C. T. Gaskins18
9
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Department of Animal Sciences, Washington State University, Pullman 99164-6351 14
15 1 Correspondence: 135 Clark Hall (phone 509-335-6416; fax 509-335-4246; e-16
mail:[email protected]). 17
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Page 1 of 38 Journal of Animal Science
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Direct Fame synthesis 2
ABSTRACT: A simplified protocol to obtain fatty acid methyl esters (FAME) directly 24
from fresh tissue, oils, or feedstuffs, without prior organic solvent extraction is presented. 25
With this protocol, FAME synthesis is conducted in the presence of up to 33% water. 26
Wet tissues, or other samples, are permeabilized and hydrolyzed for 1.5 hr at 55 °C in 1N 27
KOH in methanol containing C13:0 as internal standard. The KOH is neutralized and the 28
free fatty acids are methylated by H2SO4 catalysis for 1.5 hr at 55 °C. Hexane is then 29
added to the reaction tube, which is vortex-mixed and centrifuged. The hexane is pipetted 30
into a GC vial for subsequent gas chromatography. All reactions are conducted in a single 31
screw cap Pyrex tube for convenience. The method meets a number of criteria for fatty 32
acid analysis including not isomerizing conjugated linoleic acid (CLA) or introducing 33
fatty acid artifacts. It is applicable to fresh, frozen, or lyopholyzed tissue samples, in 34
addition to oils, waxes and feedstuffs. The method saves time, effort and is economical 35
when compared to other methods. Its unique performance, including easy sample 36
preparation, is achieved because water is included in the FAME reaction mixtures rather 37
than eliminated. 38
39
Key words: fatty acid analysis, FAME synthesis, longissimus muscle, conjugated linoleic 40
acid (CLA), fish oil, feedstuffs 41
42
Introduction 43
44
The analysis of fatty acids has become increasingly important because more people 45
have become aware of their nutritional and health implications. Because of this, a method 46
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Direct Fame synthesis 3
for analyzing fatty acids that provides both rapid and reliable results is of great value. 47
Many methods are currently used to analyze fatty acids (e.g., Morrison and Smith, 1964; 48
Sukhija and Palmquist, 1988; Kazala et al., 1999; Mir et al., 2002; Nuernberg et al., 2002; 49
Budge and Iverson, 2003; Cooper et al., 2004). These methods, however, are not 50
necessarily convenient, nor direct, and often must be optimized for reaction conditions, 51
including the catalyst and the temperature (Lewis et al., 2000; Park et al. 2002; Shahin et 52
al., 2003). On the other hand, the ideal method, as noted by Palmquist and Jenkins 53
(2003) in discussing challenges encountered in developing fatty acid methods, would 54
determine the total fatty acid concentration in tissues, oils, and feed samples by 55
converting fatty acid salts, as well as the acyl components in all lipid classes such as 56
triacylglycerols, phospholipids, sphingolipids, and waxes, to methyl esters using a simple 57
direct one-step esterification procedure. 58
In this paper we present a method that is established upon a surprising conception, i.e., 59
we add water to the fatty acid methyl ester (FAME) synthesis reagents. Until now, FAME 60
synthesis methods have rigorously avoided water as a matter of standard procedure. 61
However by adding water, the dynamics of sample preparation and methyl ester 62
formation can be revisited and the ideal outcome of FAME synthesis discussed by 63
Palmquest and Jenkins (2003) mentioned above becomes possible. Although the method 64
described herein requires two steps, it does so in one reaction tube. 65
By introducing water into FAME synthesis, the drudgery of special sample 66
preparation, e.g., freeze-drying, is no longer necessary. A sample is analyzed in the state 67
it is obtained. Thus, for example, meat and milk samples can be analyzed directly without 68
any prior dehydration step. The presence of water in the reagents allows for tissue sample 69
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Direct Fame synthesis 4
dissolution and facilitates the total extraction of fatty acids not possible in the absence of 70
water. Importantly, water does not interfere with the methylation of any fatty acid. The 71
method uses familiar and economical chemicals, namely, methanol, potassium hydroxide, 72
and sulfuric acid, but uses these methylating reagents in the presence of water. As a result 73
of these characteristics, the direct FAME synthesis method is convenient, reliable and 74
efficient. 75
In short, the objective of this paper is to develop a method to directly methylate fatty 76
acids from muscle tissue, oils, and feedstuffs in aqueous solution. 77
78
Materials and Methods 79
80
Methylation Agents and Sample Selection 81
82
To assess certain features of our method, we compared direct FAME synthesis to two 83
methylating agents routinely used for FAME synthesis, namely, the base catalyst sodium 84
methoxide and the acid catalyst boron trifluoride. Samples used with sodium methoxide 85
and boron trifluoride did not contain water, as this would compromise the performance of 86
these two methylating agents. The direct FAME synthesis method, however, always 87
contained water, even if the sample did not, because its reagents contained water, i.e., the 88
direct FAME synthesis reaction mixture contained 13.2% water due to the water in the 89
potassium hydroxide and sulfuric acid reagents. 90
The samples we used in this manuscript were chosen for distinct reasons. The Supelco 91
fatty acid standard mixture was chosen because it contained short and long chain 92
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Direct Fame synthesis 5
saturated, monounsaturated, and polyunsaturated fatty acids in defined amounts and thus 93
served as a primary test of the feasibility of our method. Fish oil was chosen because it is 94
an important source of the long chain polyunsaturated omega-3 esterified 95
eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic (DHA) fatty 96
acids. Conjugated linoleic acid (CLA), as the free acid, was chosen because it is of 97
medical importance as perhaps the only fatty acid that can directly inhibit cancer in 98
animal models (Belury, 2002) and because current FAME synthesis methods often cause 99
undesirable isomerizations of this fatty acid (Kramer et al., 1997). Beef longissimus 100
muscle was chosen because of our special interest in beef fatty acids and it serves as a 101
direct test of the ability of direct FAME synthesis to extract and methylate all of the fatty 102
acids present in meat tissue. To address the problem of refractory samples, we included 103
wax esters, cholesteryl lipid derivatives and alkyl methane sulfonates, as these are 104
difficult fatty acid derivatives to analyze (Palmquist and Jenkins, 2003). Finally, as a 105
concluding demonstration of the versatility of the direct FAME synthesis method, we 106
included a variety of oils, feedstuffs and foods. 107
108
Materials 109
110
Hexane (OmniSolv) was purchased from EM Science, Cherry Hill, NY. Absolute 111
methanol and potassium hydroxide were obtained from J.T. Baker Chemical Co., 112
Phillipsburg, NJ. Chloroform and sulfuric acid were purchased from Fisher Scientific, 113
Tustin, CA. Sodium methoxide and boron trifluoride-methanol were obtained from 114
Sigma-Aldrich, St. Louis, MO. The Supelco standard FAME mixture (47885-U) was 115
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obtained from Supelco, Bellefonte, PA. Spring Valley fish oil capsules were distributed 116
by Leiner Health Products, Carson, CA. Tonalin 1000-CLA (conjugated linoleic acid) 117
capsules were obtained from Nature Bounty, Bohemia, NY. All other fatty acid standards 118
were purchased from Nu-Chek Prep. Inc., Elysian, MN. Beef longissimus muscle 119
samples (n=20) were obtained from department owned animals processed at an abattoir. 120
Nuts and sundry food items were purchased from local grocery stores. Coffee bean 121
grinders were purchased from Mr. Coffee, Inc., Cleveland, OH. Pyrex screw-cap culture 122
tubes (16 x 125 mm) were obtained from Corning Laboratory Science Company, 123
Corning, NY. The Tekmar VXR-10 multi-tube vortex was purchased from Jenke and 124
Kunkel, West Germany. 125
126
Folch Extraction of Fatty Acids from Longissimus Muscle 127
128
Longissimus muscle (1g) was extracted with chloroform/methanol (2:1, vol:vol), 129
containing C13:0 as internal standard, according to the method of Folch et al.(1956), 130
using a Brinkmann polytron at room temperature. The extraction mixture was then 131
filtered through a glass scintered filter and replicate aliquots were pipetted into a 16 x 125 132
mm screw-cap Pyrex culture tube and washed with 0.02% aqueous CaCl2. The organic 133
phase was dried with Na2SO4 and K2CO3 (10:1, wt:wt) and the solvent was subsequently 134
removed under nitrogen at 55 °C. 135
136
FAME Synthesis with Sodium Methoxide or Boron Trifluoride 137
138
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Freeze-dried tissue samples were uniformly distributed by grinding for 10 -15 sec in a 139
room-temperature coffee bean grinder. Samples of freeze-dried tissue (0.25g), or oils (20 140
µl) were placed into a 16 x 125 mm screw- cap Pyrex culture tube to which 1.0 ml methyl 141
C13:0 internal standard (0.5mg methyl C13:0/ml methanol) was added. Two ml of 142
sodium methoxide (0.5 M) or 2 ml of boron trifluoride in methanol (14%, wt/vol) were 143
added to the Pyrex tubes containing the samples. The tubes were incubated in a 55 °C144
water bath for 1.5 h with vigorous 5 sec hand-shaking every 20 min. Two ml of a 145
saturated solution of NaHCO3 and 3 ml hexane were then added and the tubes were 146
vortex-mixed. After centrifugation, the hexane layer containing the FAME was placed 147
into a GC vial. The vial was capped and placed at –20 °C until GC analysis. 148
149
Direct FAME Synthesis 150
151
Samples were uniformly distributed by grinding for 10 -15 sec in a room-temperature 152
coffee bean grinder. Short grinding times minimize smearing fat on the grinder container 153
walls. Samples could be processed in the state obtained, e.g., wet, dry, freeze-dried, or 154
semi-frozen. Samples (0.5g wet, dry, or semi-frozen sample), (0.25g freeze-dried 155
sample), or oils (20 µl) were placed into a 16 x 125 mm screw-cap Pyrex culture tube to 156
which 1.0 ml C13:0 internal standard (0.5mg C13:0/ml methanol), 0.7 ml 10 N KOH in 157
water, and 5.3 ml methanol were added. The tube was incubated in a 55 °C water bath for 158
1.5 h with vigorous 5 sec hand-shaking every 20 min to properly permeate, dissolve and 159
hydrolyze the sample. After cooling below room temperature in a cold tap water bath, 160
0.58 ml of 24 N H2SO4 in water was added. The tube was mixed by inversion, and with 161
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precipitated K2SO4 present, was incubated again in a 55 °C water bath for 1.5 h with 5 162
sec hand-shaking every 20 min. After FAME synthesis, the tube was cooled in a cold tap 163
water bath. Three ml of hexane was added and the tube was vortex-mixed for 5 min on a 164
multi-tube vortex. The tube was centrifuged for 5 min in a tabletop centrifuge and the 165
hexane layer, containing the FAME, was placed into a gas chromatography (GC) vial. 166
The vial was capped and placed at –20 °C until GC analysis. 167
168
Gas Chromatography 169
170
The fatty acid composition of the FAME was determined by capillary gas 171
chromatography on a SPTM-2560, 100 m x 0.25 mm x 0.20 µm capillary column 172
(Supelco, Bellefonte, PA) installed on a Hewlett Packard 5890 gas chromatograph 173
equipped with a Hewlett Packard 3396 Series II integrator and 7673 controller, a flame 174
ionization detector and split injection. Initial oven temperature was 140 °C, held for 5 175
min, subsequently increased to 240 °C at a rate or 4 °C min-1, and then held for 20 min. 176
Helium was used as the carrier gas at a flow rate of 0.5 ml min-1 and column head 177
pressure was 280 kPa. Both the injector and detector were set at 260 °C. The split ratio 178
was 30:1. Fatty acids were identified by comparing their retention times with fatty acid 179
methyl standards described under Materials. 180
181
Statistical Analysis 182
183
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Duplicate gas chromatograph results were averaged for animal and methylation 184
method. ANOVA of beef longissimus muscle FAME was calculated using Proc GLM of 185
SAS (SAS Institute, Cary, NC) using a model with methylation method as the treatments 186
and animal as a blocking factor in a randomized complete block design. When the F-ratio 187
for the methylation methods was significant, Student’s t-test was used to make pairwise 188
comparisons among the means. 189
190
Results 191
192
Sodium Methoxide, Boron Trifluoride, and Direct FAME Synthesis Methods Applied to a 193
Supelco Standard FAME Mixture 194
195
For an initial FAME synthesis analysis, we used a Supelco standard FAME mixture. 196
This standard mixture contained FAME in a defined ratio, and consisted of both short and 197
long chain saturated, monounsaturated, and polyunsaturated fatty acids. In order to 198
broadly assess certain features of our method, we compared the results of direct FAME 199
synthesis to those of the base catalyst sodium methoxide and the acid catalyst boron 200
trifluoride. 201
Direct FAME synthesis, as described in Materials and Methods, is a two-step 202
procedure. In the first step, sample fatty acid esters are hydrolyzed to free fatty acids and 203
in the second step the free fatty acids are converted to FAME. When the first step of 204
direct FAME synthesis was applied to the Supelco standard FAME mixture, the esters 205
were hydrolyzed to free fatty acids that were not volatile enough to enter the GC column. 206
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These results, i.e., the absence of fatty acid peaks, provided formal evidence that the first 207
step in direct FAME synthesis completely hydrolyzed the Supelco standard FAME to free 208
fatty acids which was the desired general prerequisite for the subsequent methylation step 209
of direct FAME synthesis (data not shown). 210
When the second step of direct FAME synthesis, the methylation step, was applied to 211
the Supelco free fatty acids produced by the first step, the results shown in Table 1 were 212
obtained. All of the GC peaks present in the original Supelco standard mixture were 213
again observed, as can be seen by comparing the fatty acids of direct FAME synthesis to 214
those of the Supelco mix. When presented with a FAME sample, as in this experiment, 215
both sodium methoxide and boron trifluoride likewise gave the same FAME values 216
present in the original Supelco mix (Table 1). 217
Fatty acid artifacts, e.g., the conversion of CLA to other isomers, are a concern in fatty 218
acid analysis (Kramer et al. 1997; Park et al., 2002; Shahin et al., 2003). Since direct 219
FAME synthesis, sodium methoxide, or boron trifluoride did not generate new fatty acid 220
peaks, no fatty acid artifacts were created in the Supelco standard FAME mixture. 221
222
The Methods of Sodium Methoxide, Boron Trifluoride, and Direct FAME Synthesis 223
Applied to Fish Oil 224
225
A comparison was made between the base catalyst sodium methoxide, the acid 226
catalyst boron trifluoride, and direct synthesis, on FAME production from fish oil 227
commercially obtained as a human nutritional supplement. The results are shown in 228
Table 2. 229
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Direct Fame synthesis 11
Twenty fatty acids were identified in the fish oil sample. Direct FAME synthesis 230
recovered more total amount of fatty acid than did either of the other two methods as 231
would be expected if direct FAME synthesis generated methyl esters of all the fatty acids 232
in the sample. In comparison, base catalysis with sodium methoxide methylated esterified 233
fatty acids, but not free fatty acids (Kramer et al., 1997), whereas, acid catalysis by boron 234
trifluoride should have methylated all fatty acids, including esterified, unesterified, and 235
those in salt form (Carrapiso and Garcia, 2000). 236
In analyzing the total fatty acids methylated, direct FAME synthesis converted 22% 237
more fatty acids to FAME than did sodium methoxide and 14% more than did boron 238
trifluoride indicating that there must be groups of fatty acids present that the latter two 239
methods did not recognize. Such limitations with these two reagents have been 240
previously noted (Kramer et al., 1997; Christie, 2003). The direct FAME synthesis 241
method apparently methylates all of the fatty acids present, as confirmed by the LECO 242
Fat Extractor (see Independent Assessment of Direct FAME Synthesis Efficiency), which 243
explains why the direct FAME synthesis recoveries were higher than the other two 244
methods. 245
When the peak areas were expressed as % of total fatty acid (% FA, wt/wt) present by 246
each method, the % FAs were similar for all three methods even though total recovery 247
among the three methods was somewhat different. This indicates that the fatty acids not 248
methylated by sodium methoxide or boron trifluoride were present in similar ratios for all 249
of the fatty acids present. The results with sodium methoxide, which does not methylate 250
free fatty acids, indicates that because it methylated only 82% of the total fatty acids 251
present the other 18% of the fatty acids present may have been free fatty acids. 252
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253
Conjugated Linoleic Acid Analysis Using Sodium Methoxide, Boron Trifluoride or Direct 254
Synthesis 255
256
Table 3 presents the results obtained from an analysis of commercial CLA capsules 257
using sodium methoxide, boron trifluoride and direct synthesis. The CLA capsules 258
contained the two important CLA isomers C18:2c9,t11 and C18:2t10,c12, and also 259
palmitic (C 16:0), stearic (C18:0), oleic (C18:1n9), and linoleic (C18:2n6) fatty acids. 260
Most notable, was the difference in the results obtained with sodium methoxide 261
compared to that obtained with boron trifluoride and direct FAME synthesis. Only 0.4% 262
of the fatty acids present were methylated by sodium methoxide, indicating that they 263
were virtually all free fatty acids, and not esterified. As noted previously, sodium 264
methoxide methylates esterified fatty acids, but not free fatty acids (Kramer et al., 1997). 265
This example serves as a caution that a researcher who uses sodium methoxide, or 266
alkaline catalysts in general, is at great risk of missing fatty acids, whereas, direct FAME 267
synthesis methylates all of the fatty acids present. 268
Boron trifluoride and direct FAME synthesis gave essentially the same results for the 269
fatty acids present in the CLA capsules. Once again, the direct FAME synthesis method 270
did not generate fatty acid artifacts, including CLA artifacts, as all of the peaks were 271
essentially identical to those of the boron trifluoride method. The absence of CLA 272
artifacts confirmed the work of Park et al. (2002), who used similar H2SO4 conditions on 273
CLA samples as was used with direct FAME synthesis, but Park et al. (2002) did not 274
have water present. 275
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276
The Analysis of Beef Longissimus Muscle Using Sodium Methoxide, Boron Trifluoride or 277
Direct FAME Synthesis 278
279
The analysis of freeze-dried beef longissimus muscle fatty acids using sodium 280
methoxide, boron trifluoride and direct FAME synthesis is presented in Table 4. Once 281
again, there were striking differences among the three methods. Direct FAME synthesis 282
recovered more (P< 0.01) fatty acids than did sodium methoxide and much more than did 283
boron trifluoride. Since most of the fatty acids were esterified in longissimus muscle, as 284
opposed to unesterified in the CLA capsule (Table 3), it was not surprising that sodium 285
methoxide performed much better in FAME synthesis of this sample, although it 286
methylated only 78% of the fatty acids present compared to direct FAME synthesis. This 287
sample also shows that boron trifluoride performed much better with the free fatty acids 288
in the CLA sample (Table 3) than with the esterified fatty acids in muscle tissue. This 289
latter result was surprising because boron trifluoride can methylate all families of fatty 290
acids (Carrapiso and Garcia, 2000). Boron trifluoride methylated all of the different fatty 291
acids present, because the same peaks were present with BF3 as with direct FAME 292
synthesis, but it did not do so quantitatively. It is unclear at this time why boron 293
trifluoride gave such poor results. It can be mentioned that Bolte et al. (2002) reported 294
satisfactory FAME synthesis results using boron trifluoride on freeze-dried lamb muscle 295
tissue fatty acids by using much higher temperature and more concentrated effort, i.e., by 296
incubating at 80°C and vortex-mixing two to three times/min. However, our results do 297
not seem to be due to the fact that boron trifluoride cannot permeate the meat sample, 298
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because similar results were observed when using a chloroform:methanol extract 299
according to the method of Folch et al. (1956), where extraction of fatty acids by boron 300
trifluoride would no longer be an issue (data not shown). Apparently, and unexpectedly, 301
there are fatty acid structures in beef longissimus muscle that can be easily methylated by 302
direct FAME synthesis but not by boron trifluoride. 303
When expressed as % FA, sodium methoxide and direct FAME synthesis were quite 304
similar in their results, but boron trifluoride was different (P<0.01), and in this latter case 305
(BF3) the % FA values were higher when the concentration of fatty acid was lower. This 306
difference could be explained by the fact that boron trifluoride methylated only 46% of 307
the fatty acids present in longissimus muscle than did direct FAME synthesis, and did so 308
preferentially. With boron trifluoride, long chain unsaturated fatty acids appeared to be 309
methylated more efficiently than short chain or saturated fatty acids, whereas, direct 310
FAME synthesis methylated fatty acids without bias to chain length or structure. As a 311
result, direct FAME synthesis consistently methylated more fatty acid, averaging 1.3 312
times that of sodium methoxide and 2.2 times that of boron trifluoride. 313
314
Independent Assessment of Direct FAME Synthesis Efficiency 315
316
To determine if the direct FAME synthesis could extract all of the fatty acids present 317
in beef longissimus muscle, we independently compared it to the LECO TFE2000 Fat 318
Extractor (LECO Corporation, St. Joseph,MI). These results are shown in Table 5. For 319
the analysis of this experiment, wet tissue and freeze dried tissue was corrected to a dry 320
matter basis. Direct FAME synthesis, whether applied to dry or wet muscle tissue, 321
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extracted all of the fatty acids present when compared to the LECO TFE200 Fat 322
Extractor, assuming that the LECO values should be 6-9 % higher since the LECO values 323
also contain glycerol and cholesterol. In this respect, direct synthesis gives a truer value 324
of fat content then does the LECO extractor, which does not differentiate fatty acids from 325
total lipid. 326
327
Direct FAME Synthesis Applied to Wax Esters, Cholesteryl Derivatives, and Fatty Acid 328
Salts 329
330
The ideal FAME synthesis method would be able to analyze fatty acids from samples 331
of wax esters, cholesteryl lipid derivatives, and alkyl methane sulfonates (Palmquist and 332
Jenkins, 2003). We applied the direct FAME synthesis method to such families of fatty 333
acids and the results are shown in Table 6. Direct FAME synthesis was able to identify 334
the fatty acids in wax esters as represented by palmityl stearate (saturated series) and 335
stearyl linoleate (unsaturated series), cholestery lipid derivatives as represented by 336
cholesteryl oleate, and fatty acid salts, as represented by oleyl methane sulfonate. As the 337
second component in each of these compounds was a fatty alcohol, and not a fatty acid, it 338
was not converted to the FAME. This is as it should be, since we were analyzing strictly 339
for fatty acid components. Depending on the concentration of these very hydrophobic 340
families of fatty acids, full quantification might require more shaking and a longer 341
incubation time during the first step of direct FAME synthesis. 342
343
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Direct Fame synthesis 16
Effect of Water Content on FAME Production by Direct FAME Synthesis 344
345
It is of great interest to know what the limiting concentration of water might be for the 346
direct FAME synthesis method. There has to be such a limit, for no other reason than the 347
fact there has to be a certain concentration of methylating reagents. In Figure 1, we show 348
the effect of water concentration on the direct FAME synthesis method. As the 349
percentage of water was increased, the total amount of fatty acids methylated decreased 350
(data not shown), but this was easily corrected for by the internal standard. Most 351
importantly, the percentage of each fatty acid remained constant up to 33% water. Only 352
above 33% water do the FAME production results become problematic. In comparison, 353
our reagents, without any sample present, constitute only 13% water in a final reaction 354
volume of 7.58 ml. Thus, from a practical standpoint, one can replace 1.5 ml of MeOH 355
with a 1.5 ml aqueous sample for a final concentration of 33% water. For example, using 356
the protocol as given, 1.5 ml of milk can be analyzed directly by our method. 357
358
The Versatility of Direct FAME Synthesis 359
360
The ideal method for the analysis of fatty acids would be applicable to any sample 361
whose fatty acid content was desired. With this in mind, direct FAME synthesis was 362
applied to various products and the results are shown in Figures 2 and 3. We evaluated a 363
number of oil sources including fish, Canola, and virgin olive; a number of nuts including 364
almond, cashew, peanut, sunflower, and walnut; a couple of feedstuffs, including pelleted 365
sheep concentrate and alfalfa; and foodstuffs including beef longissimus muscle, butter, 366
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Direct Fame synthesis 17
cheese, Crisco, margarine, Miracle Whip, and Slim Fast. The direct FAME synthesis 367
method readily generated a FAME profile for all of these different samples. 368
When % FA was plotted against retention time and presented as in Figures 2 and 3, it 369
was instantly seen that each sample has its own ‘fatty acid print’. Most samples were 370
dominated by 1 to 3 fatty acids, i.e., 1 to 3 fatty acids accounted for 85%, or more, of the 371
total fatty acid composition of the sample. When the graphs were arrayed as they were in 372
these two figures, it was easy to visually compare one sample to another, e.g., the 373
vegetable oil product Crisco contained 25% oleic and 25% linoleic fatty acids, while 374
olive oil contained 70% oleic acid; beef longissimus muscle contained 40% oleic and 375
25% palmitic acids, while Slim Fast contained 70% oleic acid of the total fatty acids 376
present. The dairy products, butter and Kraft American processed cheese, had very 377
similar fatty acid profiles. The fatty acid composition of the samples presented in Figures 378
2 and 3, determined by direct FAME synthesis, are in very good agreement with the fatty 379
acid tabulations in handbooks (Watt, 1975; McCance, 2002). 380
381
Discussion 382
383
An evaluation of numerous aspects in the isolation, separation, identification and 384
structural analysis of lipids was made by Christie (2003). Among his comments on the 385
various methods of FAME synthesis, he pointed out that care should be taken in the 386
evaporation of solvents as appreciable amounts of esters up to C14 can be lost if this step 387
is performed carelessly. He stated that the vigorous use of nitrogen to evaporate solvents 388
must be avoided. In direct synthesis we eliminate solvent evaporation completely because 389
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Direct Fame synthesis 18
we do not use any prior organic solvent extraction. Christie (2003) also reminded 390
researchers that boron trifluoride in methanol has a limited shelf life, even when 391
refrigerated, and the use of old or too concentrated solutions often resulted in the 392
production of artifacts and the loss of appreciable amounts of polyunsaturated fatty acids 393
by addition of methanol across the double bonds. On the other hand, Christie (2003) 394
recommended sulfuric acid in methanol, the principle of which we have incorporated into 395
direct synthesis. Others, using sulfuric acid in methanol, have shown that it does not 396
produce CLA artifacts (Park et al., 2002). Additionally, we have used C13:0 as internal 397
standard, rather than some other odd chain fatty acid such as C19:0 because it is readily 398
soluble in methanol, a major component of our initial reagents. In principle, any fatty 399
acid may be used as internal standard as long as all of the fatty acids in the sample are 400
methylated. 401
In our method of direct FAME synthesis we introduced water into fatty acid analysis. 402
In fact, water defines our method, and stands in definite contrast to all other FAME 403
synthesis methods. Other researchers, including those who created direct methods for 404
fatty acid analysis, took special care to avoid water. Even exposure to ambient air was 405
minimized in order to avoid moisture uptake by dry muscle tissue (Murrieta et al., 2003). 406
When water cannot be used, sample preparation was no longer convenient and hydrolysis 407
of fatty acids, cholesteryl esters, and waxes, the first step of our method, was not 408
possible. 409
Again, the principle behind the direct FAME synthesis method was to dissolve, or 410
thoroughly permeate a sample, for example, beef longissimus muscle, and in the process 411
hydrolyze fatty acid structures so they could be directly methylated without any prior 412
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organic solvent extraction. Hydrolysis, of course, requires water as does solubilization 413
and permeation. KOH in methanol alone did not solubilize tissue properly, and H2SO4 in 414
methanol precipitated tissue (data not shown). In a typical assay, even without wet tissue, 415
the first step of direct FAME synthesis contained 10% water, while the second, and final 416
step, contained 13% water. 417
Even though certain fatty acids have limited solubility in water/methanol, hydrolysis 418
of fatty acid esters still occurs at the water/methanol:lipid interface and can, in fact, be 419
accounted for by the internal standard. For concentrated fat solutions, and waxes, the 420
hydrolysis step might take longer than 1.5 h at 55 º C to complete. All of our results show 421
the efficacy of the direct FAME synthesis method, which allows up to 33% water content 422
(e.g., see Figure 1). Again, KOH in MeOH alone could not solubilize and permeate 423
tissues as well as when water was added, and H2SO4 in MeOH precipitated tissues, rather 424
than solubilized them. 425
Of further interest were the results obtained by direct FAME synthesis when 426
compared, in various situations, to the sodium methoxide and boron trifluoride methods. 427
Most striking was the case of CLA analysis (Table 3) where sodium methoxide did not 428
methylate any CLA in a capsule full of it. Similarly, but not so pronounced, was the fact 429
that boron trifluoride methylated only 46% of the fatty acids present in beef longissimus 430
muscle (Table 4). In the latter instance, expressing the results as % FA could mask the 431
inadequacies of the method, but at closer examination this too would fail. If a method 432
results in a differential extraction and synthesis of FAME, then at some point the % FA 433
will be wrong. Such an example can be found with the boron trifluoride results in Table 434
4. The concentration of C20:4n6 in beef longissimus muscle was 1.6 % with the boron 435
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trifluoride method, but only 0.9% with the direct FAME synthesis method. This two fold 436
discrepancy was accounted for by the fact that boron trifluoride differentially methylated 437
a higher percentage of C20:4n6 than it did of other fatty acids present. Direct FAME 438
synthesis provided the most accurate values because it methylated all of the fatty acids 439
present in beef longissimus muscle (Table 4) as verified by an independent analysis with 440
the LECO TFE2000 Fat Extractor (Table 5). 441
Finally, direct FAME synthesis is convenient. Since water is part of the method, and 442
not antagonistic to it, sample preparation is rapid; one only weighs-out or pipets the 443
sample into a Pyrex tube and then conducts the direct FAME synthesis. Gone is the 444
preparation time it takes to lyophilize a sample (usually days), or the prior organic 445
solvent extractions and nitrogen evaporations (usually hours) that are required to 446
eliminate water in the other fatty acid methods. 447
In summary, a simplified protocol was developed to obtain fatty acid methyl esters 448
(FAME) from any sample. The method consists of two steps, conducted in a single 449
reaction tube. The protocol relies on the presence of water, which heretofore, had been 450
rigorously and tediously eliminated in FAME synthesis methods. 451
452
Literature Cited 453
454
Belury, M. A. 2002. Inhibition of carcinogenesis by conjugated linoleic acid: potential 455
mechanisms of action. J. Nutr. 132:2995-2998. 456
Bolte, M. R., R. W. Hess, W. J. Means, G. E. Moss, and D. C. Rule. 2002. Feeding lambs 457
high-oleate or high-linoleate safflower seeds differentially influences carcass fatty 458
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acid composition. J. Anim. Sci. 80:609-616. 459
Budge, S. M., and S. J. Iverson. 2003. Quantitative analysis of fatty acid precursors in 460
marine samples: direct conversion of wax ester alcohols and dimethylacetals to 461
FAMEs. J. Lipid Res. 44:1802-1807. 462
Carrapiso, A. I., and C. Garcia. 2000. Development in lipid analysis: some new extraction 463
techniques and in situ trans esterification. Lipids 35:1167-1177. 464
Christie, W. W. 2003. Lipid Analysis: Isolation, Separation, Identification and Structural 465
Analysis of Lipids, Third edition. The Oily Press, Bridgwater, England. 466
Cooper, S. L., L. A. Sinclair, R, G. Wilkinson, K. G. Hallett, M. Enser, and J. D. Wood. 467
2004. Manipulation of the n-3 polysaturated fatty acid content of muscle and adipose 468
tissue in lambs. J. Anim. Sci. 82:1461-1470. 469
Folch, J., M. Lees, and G. H. Sloane-Stanley. 1956. A simple method for the isolation 470
and purification of total lipids from animal tissue. J. Biol. Chem. 226:497-509. 471
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Weselake. 1999. Relationship of fatty acid composition to intramuscular fat content in 473
beef from crossbred Wagyu cattle. J. Anim. Sci. 77:1717-1725. 474
Kramer, J. K. G., V. Fellner, M. E. R. Dugan, F. D. Sauer, M. M. Mossoba, and M. P. 475
Yurawecz. 1997. Evaluating acid and base catalysts in the methylation of milk and 476
rumen fatty acids with special emphasis on conjugated dienes and total trans fatty 477
acids. Lipids 32:1219-1228. 478
Lewis, T., P. D. Nichols, and T. A. McMeekin. 2000. Evaluation of extraction methods 479
for recovery of fatty acids from lipid-producing microheterotrophs. J. Microb. 480
Methods 43:107-116. 481
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McCance, R. A. 2002. McCance and Widdowson’s: The composition of foods. Royal 482
Society of Chemistry, Cambridge, Great Britain. 483
Mir, P. S., Z. Mir, P. S. Kuber, C. T. Gaskins, E. L. Martin, M. V. Dodson, J. A. Elias 484
Calles, K. A. Johnson, J. R. Busboom, A. J. Wood, G. J. Pittenger, and J. J. Reeves. 485
2002. Growth, carcass characteristics, muscle conjugated linoleic acid (CLA) content, 486
and response to intravenous glucose challenge in high percentage Wagyu, Wagyu X 487
Limousin, and Limousin steers fed sunflower oil-containing diets. J. Anim. Sci. 488
80:2996-3004. 489
Morrison, W. R., and L. M. Smith. 1964. Preparation of fatty acid methyl esters and 490
dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res. 5:600-608. 491
Murrieta, C. M., B. W. Hess, and D. C. Rule. 2003. Comparison of acidic and alkaline 492
catalysts for preparation of fatty acid methyl esters from ovine muscle with emphasis 493
on conjugated linoleic acid. Meat Sci. 65:523-529. 494
Nuernberg, K., G. Nuernberg, K. Ender, S. Lorenz, K. Winkler, R. Rickert, and H. 495
Steinhart. 2002. N-3 fatty acids and conjugated linoleic acids of longissimus muscle in 496
Beef cattle. Eur. J. Lipid Sci. Technol. 104:463-471. 497
Palmquist, D. L., and T. C. Jenkins. 2003. Challenges with fats and fatty acid methods. J. 498
Anim. Sci. 81:3250-3254. 499
Park, S. J., C. W. Park, S. J. Kim, J. K. Kim, Y. R. Kim, K. A. Park, J. O. Kim, and Y. L. 500
Ha. 2002. Methylation methods for the quantitative analysis of conjugated linoleic 501
acid (CLA) isomers in various lipid samples. J. Agric. Food Chem. 50:989-996. 502
Shahin, A. M., M. K. McGuire, K. L. Ritzenthaler, and T. D. Shultz. 2003. Determination 503
of c9,t11-CLA in major human plasma lipid classes using a combination of 504
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methylating methodologies. Lipids 38:793-800. 505
Sukhija, P. S., and D. L.Palmquist. 1988. Rapid method for determination of total fatty 506
acid content and composition of feedstuffs and feces. J. Agric. Food Chem. 36:1202- 507
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Department of Agriculture, Dover Publications, New York.510
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Table 1. Effect of sodium methoxide, boron trifluoride or direct FAME synthesis on a
standard Supelco FAME mixture
Supelco Mix Na Methoxide BF3 Direct synthesisFatty acid Structure mg/g SDa mg/g SDa mg/g SDa mg/g SDa
Capric C10:0 40.02 0.28 38.93 0.29 39.91 0.55 39.40 0.24
Undecanic C11:0 20.41 0.25 19.77 0.17 20.00 0.00 20.23 0.03
Lauric C12:0 39.57 0.36 38.66 0.14 39.06 0.28 39.01 0.39
Tridecanic C13:0 20.80 0.02 20.34 0.27 20.69 0.07 20.71 0.05
Myristic C14:0 41.12 0.16 39.43 0.35 40.84 0.34 40.91 0.41
Myristoleic C14:1n5 20.38 0.32 18.53 0.17 19.88 0.05 20.05 0.01
Pentadecanoic C15:0 21.54 0.05 20.62 0.39 21.09 0.25 21.16 0.22
Pentadecenoic C15:1n5 20.60 0.22 18.90 0.25 20.47 0.29 20.22 0.07
Palmitic C16:0 63.25 0.52 62.99 0.78 63.04 0.35 62.90 0.07
Palmitoleic C16:1n7 20.86 0.15 20.07 0.15 20.83 0.09 21.07 0.14
Heptadecanoic C17:0 18.66 0.55 21.42 0.21 18.62 0.08 21.23 0.36
Heptadecenoic C17:1n7 21.29 0.07 20.93 0.57 21.42 0.12 21.57 0.20
Stearic C18:0 42.68 0.25 43.72 0.64 42.66 0.52 42.97 0.35
Elaidic C18:1n9t 21.85 0.01 22.57 1.43 21.67 0.03 21.58 0.08
Oleic C18:1n9 43.06 0.27 43.78 1.37 43.58 0.26 42.70 0.08
Linolelaidic C18:2n6t 20.15 0.19 19.92 0.13 20.02 0.51 20.26 0.05
Linoleic C18:2n6 20.89 0.14 20.25 0.61 20.87 0.11 20.58 0.10
Arachidic C20:0 41.68 0.20 42.50 0.13 41.61 0.74 42.32 0.10
Gamma- Linolenic C18:3n6 19.54 0.07 18.36 0.02 19.12 0.05 19.50 0.18
Eicosenoic C20:1n9 21.06 0.11 21.08 0.01 20.62 0.06 20.87 0.15
Linolenic C18:3n3 19.84 0.25 18.69 0.15 20.33 0.25 19.92 0.19
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Heneicosanoic C21:0 21.62 0.35 21.98 0.10 21.77 0.08 21.66 0.34
Eicosadienoic C20:2 20.44 0.07 20.30 0.16 20.65 0.08 19.85 0.67
Behenic C22:0 42.20 0.08 43.27 0.66 42.18 0.04 41.91 0.16
Eicosatrienoic C20:3n6 19.49 0.03 19.36 0.00 20.08 0.12 19.26 0.06
Erucic C22:1n9 19.86 0.58 20.88 0.13 19.40 0.36 19.05 0.22
Eicosatrienoic C20:3n3 18.50 0.03 18.82 0.21 20.47 0.48 19.51 0.13
Arachidonic C20:4n6 18.52 0.46 19.38 0.07 18.94 0.06 18.71 0.22
Tricosanoic C23:0 18.54 0.49 19.25 0.07 18.86 0.05 18.67 0.20
Docosadienoic C22:2n6 19.16 0.39 19.86 0.37 19.23 0.06 19.09 0.04
Lignoceric C24:0 34.34 1.45 37.72 0.31 31.84 0.70 32.00 0.22
Eicosapentaenoic C20:5n3 19.58 2.10 17.60 0.23 22.29 0.11 23.21 0.28
Nervonic C24:1n9 17.04 0.12 18.53 0.31 16.74 0.76 16.13 0.18
Docosahexaenoic C22:6n3 11.48 0.50 11.60 0.17 11.24 0.16 11.82 0.08
Total 880.02 10.56 880.01 10.55 880.02 7.51 880.03 6.00
a SD = Standard deviation. Three Supelco FAME standard vials were pooled and then
divided into two separate samples for methylation as described in Materials and Methods.
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Table 2. Fish oil FAME synthesis by sodium methoxide, boron trifluoride or direct FAME synthesis
Na methoxide BF3 Direct synthesis
Fatty acid Structure mg/g SDa % FAb mg/g SDa % FAb mg/g SDa % FAb
Lauric C12:0 1.01 0.05 0.2 1.14 0.11 0.2 1.01 0.11 0.1
Myristic C14:0 55.74 2.93 9.0 61.53 1.80 9.2 66.69 3.33 8.8
Myristoleic C14:1n5 3.86 0.20 0.6 4.25 0.13 0.6 4.70 0.29 0.6
Palmitic C16:0 126.54 6.66 20.4 134.06 3.15 20.1 155.95 8.34 20.5
Palmitoleic C16:1n7 59.02 3.11 9.5 63.66 1.81 9.5 70.95 3.50 9.3
Heptadecanoic C17:0 3.33 0.18 0.5 3.40 0.07 0.5 4.11 0.28 0.5
Heptadecenoic C17:1n7 1.13 0.06 0.2 1.22 0.06 0.2 1.35 0.07 0.2
Stearic C18:0 25.17 1.32 4.1 25.66 0.50 3.8 31.18 1.60 4.1
Oleic C18:1n9 81.31 4.28 13.1 85.95 2.55 12.9 98.36 4.90 12.9
Linoleic C18:2n6 20.54 1.08 3.3 19.79 0.67 3.0 21.35 0.85 2.8
Arachidic C20:0 1.83 0.10 0.3 1.71 0.01 0.3 2.43 0.16 0.3
Gamma- Linolenic C18:3n6 2.70 0.14 0.4 3.11 0.06 0.5 3.27 0.18 0.4
Eicosenoic C20:1n9 10.43 0.55 1.7 10.06 0.04 1.5 12.83 0.66 1.7
Linolenic C18:3n3 6.71 0.35 1.1 7.16 0.22 1.1 8.04 0.40 1.1
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Eicosadienoic C20:2n6 21.67 1.14 3.5 23.62 0.77 3.5 25.43 1.11 3.3
Docosadienoic C22:2n6 5.57 0.29 0.9 5.85 0.19 0.9 6.71 0.33 0.9
Eicosapentaenoic C20:5n3 117.67 6.19 18.9 127.90 3.22 19.2 142.59 6.82 18.8
Nervonic C24:1n9 2.22 0.12 0.4 2.46 0.08 0.4 3.10 0.11 0.4
Docosapentaenoic C22:5n3 12.37 0.65 2.0 12.51 0.12 1.9 14.67 0.73 1.9
Docosahexaenoic C22:6n3 62.43 3.29 10.0 71.86 0.75 10.8 85.27 4.06 11.2
Total 621.23 32.70 100.0 666.89 16.13 100.0 760.00 37.63 100.0
a SD = Standard deviation. Three fish oil capsules were pooled and then divided into two separate samples for methylation as
described in Materials and Methods.b % FA = % of total fatty acids identified in sample (wt/wt).
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Table 3. Conjugated linoleic acid (CLA) FAME synthesis by sodium methoxide, boron
trifluoride or direct FAME synthesis
Na methoxide BF3 Direct synthesis
Fatty acid Structure mg/g SDa mg/g SDa mg/g SDa
Palmitic C16:0 nd b 14.82 1.35 13.37 1.22
Stearic C18:0 nd b 24.78 1.49 24.25 2.45
Oleic C18:1n9 nd b 111.97 5.46 108.78 10.24
Linoleic C18:2n6 nd b 3.32 0.15 3.25 0.34
c9,t11 CLA C18:2c9,t11 1.35 0.12 295.08 13.91 311.90 40.40
t10,c12 CLA C18:2t10,c12 1.45 0.21 289.44 11.92 308.45 37.25
Total 2.81 0.33 739.42 34.27 770.00 91.90 a SD = Standard deviation. Three CLA capsules were pooled and then divided into two
samples for methylation as described in Materials and Methods. b nd = Not detected.
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Table 4.Beef Longissimus muscle FAME synthesis by sodium methoxide, boron trifluoride or direct FAME synthesis
Na methoxide BF3 Direct synthesis
Fatty acid Structure mg/ga ± SEb % FAc mg/ga ± SEb % FAc mg/ga ± SEb % FAc
Lauric C12:0 0.21 ±0.02d 0.09d 0.17 ±0.01d 0.12e 0.24 ±0.02e 0.08d
Myristic C14:0 8.34 ±0.68e 3.46e 5.57 ±0.25d 3.90f 10.29 ±0.83f 3.32d
Myristoleic C14:1n5 2.01 ±0.14e 0.83e 1.38 ±0.08d 0.97f 2.29 ±0.21e 0.74d
Pentadecanoic C15:0 1.59 ±0.14d 0.66d 1.24 ±0.08d 0.87f 2.23 ±0.18e 0.72e
Palmitic C16:0 62.82 ±4.10e 26.07d 38.00 ±0.86d 26.61e 80.85 ±4.84f 26.08d
Palmitoleic C16:1n7 8.10 ±0.65e 3.36d 5.74 ±0.19d 4.02e 10.91 ±0.66f 3.52d
Heptadecanoic C17:0 3.89 ±0.35e 1.61e 2.03 ±0.08d 1.42d 5.05 ±0.37f 1.63e
Stearic C18:0 30.25 ±1.89e 12.56e 15.55 ±0.28d 10.89d 39.25 ±2.31f 12.66e
t-Vaccenic C18:1n11t 19.20 ±2.36e 7.97f 9.35 ±0.96d 6.55d 22.21 ±3.28e 7.16e
Oleic C18:1n9 87.10 ±5.28e 36.15e 47.60 ±1.03d 33.34d 113.91 ±6.43f 36.75e
Linoleic C18:2n6 10.70 ±0.62d 4.44d 10.50 ±0.46d 7.36e 13.98 ±0.82e 4.51d
Arachidic C20:0 0.60 ±0.06f 0.25f 0.00 ±0.00d 0.00d 0.40 ±0.05e 0.13e
Gamma- Linolenic C18:3n6 0.32 ±0.03e 0.13e 0.17 ±0.02d 0.12e 0.28 ±0.02e 0.09d
Eicosenoic C20:1n9 0.20 ±0.01e 0.08d 0.10 ±0.00d 0.07d 0.64 ±0.05f 0.21e
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Linolenic C18:3n3 0.56 ±0.04e 0.23e 0.21 ±0.01d 0.15d 0.91 ±0.05f 0.29f
c-9,t-11 CLA C18:2c9,t11 0.70 ±0.04e 0.29d 0.59 ±0.02d 0.41e 0.85 ±0.09f 0.27d
Eicosadienoic C20:2 0.25 ±0.02e 0.10e 0.17 ±0.01d 0.12e 0.20 ±0.02e 0.06d
Behenic C22:0 0.40 ±0.04f 0.17e 0.25 ±0.02e 0.18e 0.18 ±0.01d 0.06d
Eicosatrienoic C20:3n6 0.41 ±0.06e 0.17d 0.31 ±0.06d 0.22e 0.85 ±0.03f 0.28f
Arachidonic C20:4n6 2.05 ±0.07d 0.85d 2.24 ±0.20d 1.57e 2.74 ±0.09e 0.89d
Eicosapentaenoic C20:5n3 0.40 ±0.03d 0.17d 0.52 ±0.03e 0.37e 0.54 ±0.03e 0.17d
Docosatetraenoic C22:4n6 0.18 ±0.01d 0.07d 0.24 ±0.01e 0.17e 0.26 ±0.01e 0.09d
Docosapentaenoic C22:5n3 0.68 ±0.03d 0.28d 0.84 ±0.06e 0.59e 0.93 ±0.03f 0.30d
Total 240.95 ±16.67e 100.00d 142.78 ±4.72d 100.00d 310.00 ±20.40f 100.00d
a mg/g of freeze dried tissue. b SE = Standard error (n=20). c % FA = % of total fatty acids identified in sample (wt/wt). d,e,f ANOVA
analysis was determined as described in Materials and Methods. Different superscripts indicate significant difference at P < .01 for
pairwise comparisons among the methylation methods. Columns containing mg/g were compared among themselves and columns
containing %FA were compared among themselves.
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Table 5. A comparison of fatty acid concentrations obtained from beef longissimus
muscle using direct FAME synthesis or the LECO fat extractor
Condition Fatty acid (mg / g dry matter) ± SEa
FAME synthesis
Freeze dried tissueb 310.25 ± 18.23
Wet tissueb 312.37 ± 27.51
LECOc 336.59 ± 23.36 aSE = Standard error (n=12)
bSamples were analyzed by direct FAME synthesis. cLipid content was determined in freeze dried tissue by the LECO TFE2000 Fat
Extractor.
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Direct FAME synthesis 32
Table 6. Direct FAME synthesis of samples from wax esters, cholesterol lipid derivatives
and alkyl methane sulfates
Compound Acid component Ester observed
Palmityl stearate Stearic Methyl stearate
Stearyl linoleate Linoleic Methyl linoleate
Cholesteryl oleate Oleic Methyl oleate
Oleyl methane sulfonate Methane sulfonic Methyl methane sulfonate
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Figure 1.Effect of water concentration on FAME production of fish oil fatty acids and
internal standard by direct FAME synthesis.
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0
5
10
15
20
25
30
10 15 20 25 30 35 40 45 50 55Percent Water
Perc
entF
attyA
cid
C16:0C20:5n3C18:1n9C22:6n3C18:0C13:0C20:2n6C22:5n3C18:3n6
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Direct FAME synthesis 35
Figure 2. Direct FAME synthesis from samples of Supelco FAME mixture, fish oil,
Crisco, pelleted sheep concentrate, beef longissimus muscle, canola, alfalfa, Slim Fast,
and virgin olive oil. % Fatty acid = % of total fatty acid present in sample.
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Supelco FAME Mixture
0.01.02.03.04.05.06.07.0
Ret ent ion t ime ( min)
Fish Oil
02468
1012141618
10 50Retention time (min)
%Fa
ttyac
id
Crisco
0
5
10
15
20
25
30
R et ent io n t ime ( min)
Pelleted Sheep Concentrate
05
1015
2025303540
R et ent io n t ime ( min)
Beef Longissimus Muscle
010
2030
4050
R et ent io n t ime ( min)
Canola
010203040506070
R et ent io n t ime ( min)
Alfalfa
05
1015
202530
R et ent io n t ime ( min)
Slim Fast
01020304050607080
R et ent io n t ime ( min)
Virgin Olive Oil
01020304050607080
Ret ent ion t ime ( min)
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Figure 3. Direct FAME synthesis from samples of sunflower seeds, peanuts, Blue
Bonnet margarine, walnuts, almonds, butter, cashews, Miracle Whip, and Kraft American
processed cheese. % Fatty acid = % of total fatty acid present in sample.
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Sunflower
010203040506070
R et ent io n t ime ( min)
Peanut
010203040506070
R et ent io n t ime ( min)
Blue Bonnet Margarine
05
1015
20253035404550
R et ent io n t ime ( min)
Walnut
010203040506070
R et ent ion t ime ( min)
Almond
01020304050607080
R et ent io n t ime ( min)
Butter
05
1015
202530
Ret ent ion t ime ( min)
Cashew
010203040506070
Ret ent ion t ime ( min)
Miracle Whip
0102030405060
Ret ent ion t ime ( min)
Kraft Cheese
05
1015
202530
Ret ent ion t ime ( min)
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Citations
cleshttp://jas.fass.org/content/early/2007/02/12/jas.2006-491.citation#otherartiThis article has been cited by 8 HighWire-hosted articles:
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