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J. Anim. Sci. 2003. 81:3250-3254
© 2003 American Society of Animal Science

Challenges with fats and fatty acid methods

D. L. Palmquist*,1 and T. C. Jenkins{dagger}

* Department of Animal Sciences, OARDC/OSU, Wooster and and {dagger} Department of Animal and Veterinary Sciences, Clemson University


   Abstract
Top
 Abstract
Introduction
 Lipid Composition of Feedstuffs
 Determiniation of Lipid Content...
 Other Considerations
 Recommendations
 Implications
 Literature Cited
 
The content and chemical nature of lipids in feedstuffs is heterogeneous. It has long been known that ether extraction by the Weende procedure inadequately characterizes the fat content of feedstuffs, yet it remains the official method. Diethyl ether (or hexanes that are often used) extracts significant amounts of nonnutritive, nonsaponifiable lipids from forages, and often incompletely extracts lipids of nutritional value, especially fatty acids present as salts of divalent cations. Preextraction hydrolysis of insoluble fatty acid salts with acid releases these fatty acids, and this step is included in the official procedure for certain feedstuffs in the United Kingdom; however, acid hydrolysis increases analysis time and decreases precision. Acid hydrolysis also causes confusion as to the proper definition of the fat content of feedstuffs. A preferred method of fat analysis determines the total fatty acid concentration in feed samples by converting fatty acid salts, as well as the acyl components in all lipid classes, such as triacylglycerols, phospholipids, and sphingolipids, to methyl esters using a simple, direct one-step esterification procedure. Fatty acid methyl esters are then quantified by GLC, which provides information on both fatty acid quantity and profile in a single analysis. Adjustments in conditions and reagents may be necessary to overcome difficulty in quantitatively preparing esters from certain types of fatty acids and their derivatives in commercial fat supplements. After correction for glycerol content, analysis of oils by this procedure provides information on the content of nonsaponifiable material, such as chlorophyll, waxes, and indigestible polymers formed from heat- or oxidatively damaged fats. The correct description of feedstuffs for energy value of fats is the content of total fatty acids.

Key Words: Ether Extracts • Fats • Fatty Acids • Gas Chromatography


   Introduction
Top
 Abstract
 Introduction
Lipid Composition of Feedstuffs
 Determiniation of Lipid Content...
 Other Considerations
 Recommendations
 Implications
 Literature Cited
 
Increasing international trade and advances in biotechnology have caused a need for a greater amount of and more sophisticated information with regard to composition and quality of foodstuffs and feedstuffs in the market. Similarly, there is a greater understanding of nutrient utilization by livestock, which has led to the development of sophisticated computer models for feeding management and prediction of performance by domestic animals. With increasing need for accurate data, it is necessary to examine procedures by which data are obtained; this article reviews relevant procedures and presents recommendations for reporting the content of fats in feedstuffs. Hammond (2001)Go has provided a useful summary of advances in lipid analysis in the 20th century.


   Lipid Composition of Feedstuffs
Top
 Abstract
 Introduction
 Lipid Composition of Feedstuffs
Determiniation of Lipid Content...
 Other Considerations
 Recommendations
 Implications
 Literature Cited
 
The lipid content of feedstuffs varies widely, from less than 1% in some byproducts to 100% in some fat supplements (Palmquist, 1988Go; van Soest, 1994Go). Similarly, the composition of the crude lipid (ether extract) also varies widely. Triacylglycerol (90% fatty acid) is the major lipid class in rendered fats, in most cereals, and in oil seeds (>95, 2 to 8, and 18 to 45% ether extract, respectively), whereas total fatty acid in forages is often less than 50% of the ether extract (Palmquist and Jenkins, 1980Go; van Soest, 1994Go). A large part of ether extract in forages is comprised of nonsaponifiable substances (waxes, chlorophyll, cutin, etc.) (Table 1Go). The majority of lipid in forages is found in the chloroplasts and its proportion of the plant dry weight decreases as the plant matures (Hawke, 1973Go). Triacylglycerol is potentially completely metabolizable by animals, whereas the nonsaponifiable fraction has no energy value, although it may offer other desirable nutritional characteristics (e.g., fat-soluble vitamins, carotene). Glycerol (10 to 11% of the glyceride by weight) has an energy value comparable to other carbohydrates, whereas the fatty acids contribute the highly dense energy value of fats. The ether extract fraction of plants, because it contains numerous nonnutritive substances, is not a nutritionally uniform fraction, whereas fatty acids constitute a uniform fraction that can be measured and used in computer modeling to estimate the energy value of feedstuffs (Weiss, 1993Go; NRC 2001Go). It is instructive to note that the FDA, as a result of the Nutrition and Labeling Education Act of 1990, defines total fat in foodstuffs as the sum of all fatty acids obtained from a total lipid extract, expressed as triglycerides (Eller, 1999Go).


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  		Table 1. Concentration and composition of ether extract from forage leavesa
 

   Determiniation of Lipid Content of Feedstuffs
Top
 Abstract
 Introduction
 Lipid Composition of Feedstuffs
 Determiniation of Lipid Content...
Other Considerations
 Recommendations
 Implications
 Literature Cited
 
Ether Extraction
Ether extraction is the classical method for lipid determination of feedstuffs. The establishment of ether extract as a nutritive component in feedstuffs evaluation is generally attributed to Henneberg and Stohmann, working at the Weende Experiment Station in Germany circa 1860 (Flatt, 1988Go). However, the procedures described for "ether extract" in AOAC (Horwitz, 2000Go) continue to be limited, and consequently there is considerable variability in adherence to procedures for measurement of ether extract (Thiex et al., personal communication). Because "ether extract" is defined by the method used to measure it, it is crucial that methods be followed exactly (Thiex et al., 2003Go). The official AOAC method (920.39, Horwitz, 2000Go) specifies that the solvent shall be diethyl ether, yet many laboratories use petroleum ether or hexane. Hexanes are less polar, thus extracting less membrane lipid and yielding a lower value (96.7% of the diethyl ether value, Thiex et al., 2003Go); however, diethyl ether also extracts certain water-soluble components, such as urea and hexoses, thereby potentially yielding inaccurate or artificially high values. The official AOAC method is similarly vague with regard to extraction time, indicating "4 hr at condensation rate of 5 to 6 drops/sec to 16 hr at 2 to 3 drops/sec." It would be a rare laboratory that monitors drop rate precisely; thus, extraction time often becomes an unspecified variable in determination of ether extract. A recent collaborative study (Thiex et al., 2003Go) documented procedures and precision of ether extraction of feedstuffs, comparing diethyl ether, petroleum ether, hexanes, and pentanes using the Randall/Soxtec/Submersion method that is an official AOAC method for meats and meat products (991.36; Horwitz, 2000Go). The submersion method was shown to decrease required extraction time and provide increased labor efficiency compared to the Soxhlet procedure; however, great attention to procedural detail was required to achieve precision among laboratories. It was concluded that hexanes were an acceptable alternative to diethyl ether as a solvent.

Acid-Ether Extraction
Difficulties with low oil recovery from fat-filled milk powders and newly introduced calcium salts of fatty acids as a rumen-inert fat supplement in the United Kingdom led to regulatory changes in extraction procedures for oil from many compound feeds and feeds of animal origin (Sanderson, 1986Go). These included boiling the feed sample in 3N HCl, followed by washing with water before the ether extraction step. This modification yielded consistently higher oil values than ether extraction alone, thus raising another difficulty (i.e., which value is "correct" and whether the same procedures were being used for a specific feeding stuff both by analysts and salespeople; Cooke, 1986Go). The acidified ether extraction (modified Werner-Schmidt method) adapted from AOAC 954.02 (Horwitz, 2000Go) is the recommended procedure for fat analysis of calcium soap feed supplements (product bulletin, Church & Dwight Co., Inc., Princeton, NJ).

Both the merit and demerit of acid treatment before ether extraction is shown in Table 2Go (Sukhija and Palmquist, 1988Go). Extraction with petroleum ether/10% glacial acetic acid clearly increased the weight of "oil" recovered for alfalfa hay, corn silage, and low- and high-fat concentrate mixes. Fatty acid analysis of the acid extracts yielded higher values than without acid treatment, and the proportion of fatty acid in the acidified ether extract was higher for all samples except corn silage. However, acid extraction also increased nonfatty acid material (14.4 mg/g) in the corn silage sample, which may be attributable to increased extraction of nonvolatile lactic acid.


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  		Table 2. Lipid content (mg/g of DM) by Soxhlet extraction: A comparison of solvent and quantitation methodsa
 
Edmunds (1990)Go presented a comprehensive summary of the analysis of feed fats and showed that direct analysis of fatty acids was the most rapid and precise approach to determine energy value of fats. Outen et al. (1976)Go had previously documented a direct in situ extraction/methylation procedure for quantitative analysis of fatty acids in feeds, digesta, and feces. We (Sukhija and Palmquist, 1988Go) adapted and considerably simplified the procedure of Outen et al. (1976)Go for routine quantitative analysis of fatty acids in feedstuffs and feces. Numerous others have published similar procedures for specific applications (See Carrapiso and Garcia, 2000Go, for a comprehensive review).

Esterification of Fatty Acids
Our procedure (Sukhija and Palmquist, 1988Go) continues to be used essentially as described, with the exception that results are more consistent with higher concentrations of methanolic HCl–a 10% HCl solution (20 mL of acetyl chloride:100 mL of methanol) is routinely used (See Table 3Go). For high-salt samples (calcium soaps, feces) a greater volume of 10% methanolic HCl may be required. In our hands, the coefficient of variation of a range (1.5 to 11.0%) of fatty acids in feedstuffs was 3.6%.


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  		Table 3. Procedure to determine total fatty acid content of biological samples (adapted from Sukhija and Palmquist, 1988Go)
 
Conditions for extraction and in situ methylation were reviewed thoroughly by Carrapiso and Garcia (2000)Go. Acid-catalyzed methylation is commonly used because it is effective under many conditions and methylates all fatty acids, including esterified, unesterified, and those in salt form, whereas alkaline methylation only transesterifies fatty acids that are in glyceride form. Acid esterification is sometimes criticized for its potential to cause migration of conjugated double bonds in unsaturated fatty acids (Kramer et al., 1997Go). With increasing interest in cis and trans isomers of conjugated fatty acids where accurate determination of specific isomers is desired, the preferred method is to use alkaline transesterification followed by a mild acid esterification (Kramer et al., 1997Go). For determination of milk fatty acid profiles, these procedures may be adapted for transesterification with isopropanol (Wolff and Fabien, 1989Go) or with butanol (Ulberth et al., 1999Go), both of which yield uniform flame ionization detector response independent of fatty acid chain length. Thus, differential response factors to correct for chain length are not required.


   Other Considerations
Top
 Abstract
 Introduction
 Lipid Composition of Feedstuffs
 Determiniation of Lipid Content...
 Other Considerations
Recommendations
 Implications
 Literature Cited
 
Lipid Extraction
Sukhija and Palmquist (1988)Go had difficulty extracting the fatty acids in whole oilseeds quantitatively. Although they found that chloroform was superior to benzene as an oilseed extractant, neither is a desirable solvent for routine laboratory use; however, they did find that toluene substitutes suitably for benzene. Perhaps a useful aid to extracting whole oilseeds would be to apply the solvents in combination with microwaving as described by Carrapiso and Garcia (2000)Go. Heptane may be a suitable solvent for extracting feedstuffs other than oilseeds. These procedures require only small amounts of solvent, and we have had no difficulty using any of them in properly functioning laboratory hoods. For extraction of plant and animal tissues for analysis of total lipids, a hexane:isopropanol mixture (3:2, vol/vol) has been shown to be preferable to chloroform:methanol (2:1) for most applications, based on lower toxicity and cost and simpler handling of extracts (Radin, 1981Go). The acid-catalyzed methylation was shown to be efficient and quantitative for extracted lipids separated by thin layer chromatography (Sukhija and Palmquist, 1988Go) and by elution from aminopropyl bonded-phase columns (Bateman and Jenkins, 1997Go).

Gas-Liquid Chromatography Conditions
To obtain acceptable analytical precision it is necessary to have autoinjection of samples onto the GLC column. As in all quantitative GLC analyses, it is necessary to document chromatography conditions, identify peaks with appropriate standards, and determine detector responses to calculate correction factors (Christie, 1989Go). For example, acid catalysis with heating of high-sugar samples can lead to the formation of methyl levulinate that elutes just after methyl laurate (12:0) in typical GLC chromatograms (Eller, 1999Go). In our laboratory, we have observed that this can be a problem in analysis of barley, corn silage, and in highly processed commercial feedstuffs. It is probably also appropriate to reiterate that the antioxidant BHT elutes in the position of methyl myristate (14:0) on most polar GLC columns.

Internal Standards
Many fatty acids have been used as internal standards. Requirements for a suitable standard are 1) that it be unique (not present in the sample); if not unique, separate runs with and without the standard must be run; 2) it must have chemical characteristics that are similar to the unknowns (ease of esterification, GLC behavior, etc.); 3) it must be readily available; and 4) economical. Some investigators report using heptadecanoic (17:0) acid as internal standard. Because 17:0 occurs commonly in ruminant fat (1.5% in tallow), erroneous results will be obtained unless a separate GLC run without the internal standard is conducted to correct the data; therefore we recommend using 19:0.


   Recommendations
Top
 Abstract
 Introduction
 Lipid Composition of Feedstuffs
 Determiniation of Lipid Content...
 Other Considerations
 Recommendations
Implications
 Literature Cited
 
As noted above, the need in lipid analysis is to quantify the nutritionally uniform fraction of feedstuffs (i.e., total fatty acids). It is unfortunate that the recent NRC dairy (NRC, 2001Go) was required to rely on ether extract for its lipid database due to limited data for quantities of fatty acids in feedstuffs. Based on a limited data set, Allen (2000)Go suggested that fatty acid content of diets could be predicted as follows: FA = -0.98 + (1.03 x EE), r2 = 0.87; P < 0.0001; root mean square error, = 0.71; n = 18, where FA = fatty acid content of the diet (percentage of DM) and EE = ether extract content of the diet (percentage of DM). Thus, in mixed diets, fatty acid approximates the percentage of ether extract minus 1. It is necessary that investigators who report lipid analysis in their publications provide both ether extract and total fatty acid quantities in order to establish a relationship for individual feedstuffs with greater confidence.


   Implications
Top
 Abstract
 Introduction
 Lipid Composition of Feedstuffs
 Determiniation of Lipid Content...
 Other Considerations
 Recommendations
 Implications
Literature Cited
 
Hammond (2001)Go quoted the preface to E. R. Bolton’s Oils, Fats and Fatty Foods, published in the early 20th century: "In no department of analytical chemistry is greater difficulty experienced than that which deals with the examination of food products. Of these food products perhaps none presents so much diversity in natural composition as do the oils and fats. The analysis of oils and fats is both prolific of method and uncertain in result." It is unfortunate that the standard fat analysis used by the feed industry of the 21st century continues to rely on principles and methods developed in the 19th century. Hammond (2001)Go also stated later in his review that "the invention of gas chromatography is probably the single most important event of 20th century analytical science." It is time that the feed industry, regulators, and nutritionists move to apply this technology to measure the nutritionally uniform lipid fraction of feedstuffs with precision and accuracy.

1 Correspondence—palmquist.1{at}osu.edu.

Received for publication August 7, 2002. Accepted for publication November 19, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Lipid Composition of Feedstuffs
 Determiniation of Lipid Content...
 Other Considerations
 Recommendations
 Implications
 Literature Cited
 


Allen, M. S. 2000. Effects of diet on short-term regulation of feed intake by lactating dairy cattle. J. Dairy Sci. 83:1598–1624.[Abstract]

Bateman, II, H. G., and T. C. Jenkins. 1997. Method of extraction and separation by solid phase extraction of neutral lipid, free fatty acids, and polar lipid from mixed microbial cultures. J. Agric. Food Chem. 45:132–134.

Carrapiso, A. I., and C. Garcia. 2000. Development in lipid analysis: Some new extraction techniques and in situ trans esterification. Lipids 35:1167–1177.[Medline]

Christie, W. W. 1989. Gas Chromatography and Lipids: A Practical Guide. The Oily Press. P. J. Barnes & Assoc., Bridgwater, U.K.

Cooke, B. C. 1986. The implications to research and the feed compounder of the new oils and fats determination. Pages 81–86 in Recent Advances in Animal Nutrition–1986. W. Haresign and D. J. A. Cole, ed. Butterworths, London.

Edmunds, B. K. 1990. Quality control of feed fats—the implications for the feed compounder. Pages 2–8 in Proc. Int. Anim. Nutr. Symp., Natl. Renderers Assoc., Brussels, Belgium.

Eller, F. J. 1999. Interference by methyl levulinate in determination of total fat in low-fat, high-sugar products by gas chromatographic fatty and methyl ester (GC-FAME) analysis. J. Assoc. Off. Anal. Chem. Int. 82:766–769.

Flatt, W. P. 1988. Feed evaluation systems: Historical background. Pages 1–22 in World Animal Science. B. Disciplinary Approach. 4. Feed Science. E. R. Orskov, ed. Elsevier, Amsterdam.

Hammond, E. W. 2001. Lipid analysis–a 20th century success? J. Sci. Food Agric. 82:5–11.

Hawke, J. C. 1973. Lipids. Pages 213–231 in Chemistry and Biochemistry of Herbage. Vol. 1. G. W. Butler and R. W. Bailey, ed. Academic Press, New York.

Horwitz, W. 2000. Official Methods of Analysis. 4.5.01. Crude fat or ether extract in animal feed. 41.1.33. Polymers and oxidation products of heated vegetable oils. 17th ed. Assoc. Offic. Anal. Chem., Washington, DC.

Kramer, J. K. G., V. Fellner, M. E. R. Dugan, F. D. Sauer, M. M. Mossoba, and M. P. Yurawecz. 1997. Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids. Lipids 32:1219–1228.[Medline]

NRC. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Subcommittee on Dairy Cattle Nutrition. Committee. on Anim. Nutr. Board on Agric. and Nat. Res. Natl. Res. Counc. Natl. Acad. Press, Washington, DC.

Outen, G. E., D. E. Beever, and J. S. Fenlon. 1976. Direct methylation of long-chain fatty acids in feeds, digesta and faeces without prior extraction. J. Sci. Food Agric. 27:419–425.[Medline]

Palmquist, D. L. 1988. The feeding value of fats. Pages 293–311 in World Animal Science. B. Disciplinary Approach. 4. Feed Science. E. R. Orskov, ed. Elsevier, Amsterdam.

Palmquist, D. L., and T. C. Jenkins. 1980. Fat in lactation rations: Review. J. Dairy Sci. 63:1–14.

Radin, N. S. 1981. Extraction of tissue lipids with a solvent of low toxicity. Methods Enzymol. 72:5–7.[Medline]

Sanderson, P. 1986. A new method of analysis of feeding stuffs for the determination of crude oils and fats. Pages 77–80 in Recent Advances in Animal Nutrition–1986. W. Haresign and D. J. A. Cole, ed. Butterworths, London.

Sukhija, P. S., and D. L. Palmquist. 1988. Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. J. Agric. Food Chem. 36:1202–1206.

Thiex, N. J., S. Anderson, and B. Gildemeister. 2003. Crude fat extraction in feed, cereal grain and forage (Randall/Soxtec/Submersion method): A collaborative study. J. Assoc. Off. Anal. Chem. Int. 86:888–908.

Ulberth, F., R. G. Gabernig, and F. Schrammel. 1999. Flame-ionization detector response to methyl, ethyl, propyl, and butyl esters of fatty acids. J. Am. Oil Chem. Soc. 76:263–266.

van Soest, P. J. 1994. Nutritional Ecology of the Ruminant. 2nd ed. Chapter 20 in Lipids. Cornell Univ. Press, Ithaca, NY.

Weiss, W. P. 1993. Predicting energy value of feeds. J. Dairy Sci. 76:1802–1811.[Abstract]

Wolff, R. L., and R. J. Fabien. 1989. Utilisation de l’isopropanol pour l’extraction de la matiere grasse de produits laitiers et pour l’esterification subsequente des acides gras. Le Lait 69:33–46.


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