Jacqueline L Dupont. Cambridge World History of Food. Editor: Kenneth F Kiple & Kriemhild Conee Ornelas. Volume 1, Cambridge University Press, 2000.
The history of the scientific documentation of the need for fat in the diet began with the early nineteenth-century work of Michel Eugene Chevreul (Mayer and Hanson 1960). He showed that lard contained a solid fat, which he termed stearine, and a liquid fat he called elaine (later shown to be the isomer of oleine), and in 1823, this work was published in a treatise, Chemical Investigations of Fats of Animal Origin. Chevreul also crystallized potassium stearate, naming it “mother-of-pearl” and calling its acidified product “margarine” (from the Greek word for mother-of-pearl). In addition, Chevreul isolated various acids from fats and distinguished them on the basis of their melting points.
Meanwhile in 1822, Edmund Davy had reported that iodine would react with fats, and by the end of the century, work by L. H. Mills and Baron Hubl led to the procedure devised by J. J.A.Wijs in 1898 for determining a fat’s “iodine value” or “iodine number”—a measure of the extent to which a fat is unsaturated, based on its uptake of iodine. Highly saturated coconut oil, for example, has an iodine number of 8 to 10, whereas that of highly unsaturated linseed oil ranges from 170 to 202.
Phospholipids were described in 1846 by N. T. Gobley, who found that egg yolk had a substance that contained nitrogen and phosphorus in addition to glycerol and fatty acids. He named it lecithin. The nitrogenous base was shown to be choline by A. Strecker in 1868, and J. W. L. Thudichem described kephalin in 1884 (Mayer and Hanson 1960).
These early advances in chemistry and methodology were necessary before the major leap to studying the fats in nutrition could be taken. Indeed, advances in chemistry and technology have preceded key discoveries in the study of essential fatty acids and their functions throughout its history.
The Concept of Essential Fatty Acids
During the late 1920s, several groups of investigators explored questions of the nutritional value of fat that went beyond the knowledge that dietary fats provided energy and contained vitamins A and D. Among them were Herbert M. Evans and George O. Burr, who experimented with diets that were sufficiently purified to exclude fat. In three published papers (1927a; 1927b; 1928), they described a previously unknown deficiency disease that resulted from an absence of dietary fats and suggested the existence of a new vitamin, “vitamine F.” Shortly after this, at Yale University, Ava Josephine McAmis, William E. Anderson, and Lafayette B. Mendel determined that a nonsaponifiable fraction of cod liver oil delivered vitamin A—but no fat—to experimental rats, and that slightly better growth was achieved when about 20 milligrams (mg) of peanut oil was fed to the rats along with the cod liver oil fraction. Animals receiving the most peanut oil grew best. The researchers wrote, with exemplary caution, that “whether this apparent beneficial effect of a small amount of fat is due to its content of vitamin A or other vitamins, or to its acting as a vehicle for the fat-soluble vitamins, or whether fat per se is essential, is not conclusively demonstrated” (McAmis, Anderson, and Mendel 1929: 262).
At the same time, George and Mildred Burr, at the University of Minnesota, published their results from feeding a very low fat diet to rats (1929). They concluded that there was, indeed, a requirement for fat in the diet and also believed that they had discovered a new deficiency disease curable by the feeding of small amounts of unsaturated fats or pure “linolic [sic] ” acid. The following year, they coined the term “essential fatty acids” (1930).
The symptoms observed in rats that were considered indicative of dietary insufficiency included late failure of growth, kidney lesions, abnormal water consumption (because of excessive extradermal water loss), scaly skin, and necrotic tails. However, the concept of the essentiality of fatty acids was not immediately accepted. Critics pointed out that the skin lesions of rats, for example, were also seen with some B-vitamin deficiencies and thus not specific to fat deficiency. In addition, there was confusion surrounding the nature of the “fat-free” diets fed to rats because those consuming cornstarch managed some growth, whereas those on sucrose did not.
These questions were explained by the studies of Evans and Samuel Lepkovsky (at the University of California, Berkeley), who, first of all, showed that there was enough fat in cornstarch to support some growth in rats (1932a). They determined further that, if saturated fat (in this case, coconut oil) rather than carbohydrate was fed to the rats, deficiency symptoms became evident even more rapidly (1932b). In still another experiment (1932c), they isolated the fatty acid methyl esters from carcasses of rats that had been on either the fat-free or the supplemented diets, fed the esters to other rats, and discovered that the iodine numbers of fatty acids from fat-free–fed animals were higher than those from rats fed supplemented diets, thus showing that the degree of unsaturation is not a criterion of fatty acid essentiality.
The fat from the rats fed fat-free diets contained more unsaturated fatty acids than that from other rats at weaning, but the unsaturated fatty acids present did not relieve the deficiency symptoms, which was the first indication of the biosynthesis of eicosatrienoic acid. This phenomenon was later observed by Raymond Reiser and colleagues (1951) and finally explained by A. J. Fulco and J. F. Mead (1959). In the meantime, Evans, Lepkovsky, and E.A. Murphy had added both failure of reproduction and lactation in female rats (1934a, 1934b) and sterility in male rats (1934c) to the list of deficiency symptoms.
Requirement for Essential Fatty Acids
The reports of skin lesions in rats fed fat-free diets caused pediatrician Arild E. Hansen (1933) to suspect that infants suffering from eczema had an unsatisfied fat requirement. Hansen treated the condition with various oils, and shortly thereafter, Theodore Corn-bleet reported the highly successful treatment of eczema by dietary supplementation with corn oil (1935). He also noted that this supplementation brought relief to his patients with asthma.
Later, in collaboration with Hilda F. Wiese and others at the University of Texas School of Medicine in Galveston, Hansen studied essential fatty acid requirements using dogs as the experimental model (Hansen and Wiese 1951). As their methods for quantifying fatty acids progressed from fractionation of methyl esters to spectrophotometric analysis of the alkali-conjugated fatty acids (Wiese and Hansen 1953), Hansen and Wiese were able to determine serum levels of unsaturated fatty acids in both poorly nourished (Hansen and Wiese 1954) and healthy (Wiese, Gibbs, and Hansen 1954) children. This work resulted in hard evidence of a deficiency of essential fatty acids that could occur in infants; it also resulted in recommendations for dietary intake of linoleic acid based upon the serum levels of di-, tri-, and tetraenoic acids of infants fed formulas containing different sources of fat (Wiese, Hansen, and Adams 1958). The researchers concluded that the dietary linoleate needed to provide optimum serum concentrations of polyunsaturated fatty acids was about 4 percent of the total calories. This advance in understanding the biochemistry of fatty acids in serum led to the use of biochemical criteria for defining nutrient deficiency.
Interrelationships among Fatty Acids
The alkaline isomerization method for analyzing specific fatty acids had led to the deduction that arachidonic (tetraenoic) acid was formed from dietary linoleic acid and that pentaenoic and hexaenoic acids were formed from dietary linolenic acid (Rieckehoff, Holman, and Burr 1949; Widmer and Holman 1950). Moreover, studies by Ralph Holman and his group at the Hormel Institute, University of Minnesota, also determined that linoleic and linolenic acids were not interconvertible. These findings were confirmed by the use of radioisotopically labeled fatty acids (Steinberg et al. 1956), and radioisotope tracer methods were used to define the source of Evans and Lepkovsky’s (1932c) noncurative polyunsaturated fatty acid. A trienoic acid was identified by Fulco and Mead (1959) as 5,8,11-eicosatrienoic acid, which they found was derived from oleic acid.
During the 1960s, gas-liquid chromatography became the method of choice for the identification and quantification of fatty acids. A series of dose-response studies using single pure unsaturated fatty acids was conducted by Holman and his associates (Holman 1964), who developed interaction relationships which showed that the ratio of triene (eicosatrienoic acid) to tetraene (arachidonic acid) was proportional to the linoleate concentration in the diet of rats. They also determined that the diene and triene fatty acids were competitive with each other in conversions to longer-chain metabolites. For instance, 0.1 percent of energy as linolenate inhibited metabolism of linoleate by 50 percent, whereas it required 3.2 percent of linoleate to inhibit linolenate conversion by 50 percent. Interpretation of their many experiments led to the presentation of the sequence of chain elongation and desaturation of linoleic acid as 18:2 to 18:3 or 20:3; 18:3 to 20:3 to 20:4 or 22:3; 20:4 to 22:4 to 24:4 or 22:5. In the absence of linoleate and linolenate, the conversion of endogenous oleate (18:1) to 20:3 and 22:3 becomes dominant.
Nomenclature for Essential Fatty Acids
In studies prior to those of Holman and others in the 1960s, the original common names of the fatty acids were used. However, as the complexities of their double-bond configurations began to define their places in metabolism, it became necessary to establish simple and clear nomenclature. Diene, triene, monoene, and saturated, along with chain length, were no longer sufficient to describe physiologically important activities of the compounds. The locations of the double bonds were clarified, but the delta notation for the position of the double bonds was confusing when applied to chain elongation and desaturation. Holman (1964) used nomenclature based on the position of the double bond in relation to the nth (or omega) carbon. The linoleic acid family began with a double bond at the n minus 6 position; therefore, it was called the omega 6 family. The naturally occurring polyunsaturated fatty acids have methyl interrupted, rather than conjugated spacing; so linoleate is 18:2 omega 6,9. In the same pattern, linolenic acid is 18:3 omega 3,6,9. The importance of the specificity of desaturases was yet to be explained.
Importance of Human Requirements
Even though it had become accepted that linoleate was an essential nutrient, recommendations for dietary consumption were considered only for infants. Hansen, the pediatrician, agreed with Holman, the biochemist, that 0.5 to 1 percent of energy was enough, based upon keeping the triene–tetraene ratio below 0.4 (Holman 1973). By this time, the feeding of a fat-deficient diet to human subjects was not acceptable, so human studies were conducted using supplements; and, of course, diagnosis of malnourished infants also yielded findings.
The definitive proof of essential fatty acid requirement, however, came with the advent of intravenous feeding and its ability to provide total nutritional support (made possible by the method of implantation of a catheter in the superior vena cava, allowing infusion of hyperosmolar fluid) for long periods of time (Dudrick et al. 1968). Early formulas used glucose-protein hydrolysate fluid with electrolytes, minerals, and vitamins added, and reports of essential fatty acid deficiency symptoms in infants began to appear (Hall-berg, Schuberth, and Wretlind 1966). Fatty acid analyses were made when a case came to the attention of the Hormel Institute group, which reported that after 100 days of total parenteral nutrition, the infant had a triene–tetraene ratio of 18 and extreme scaliness of skin (Paulsrud et al. 1972).
The first efforts to add lipids to intravenous formulas were unsuccessful. An emulsion containing cottonseed oil proved unacceptable because of toxic reactions, and that experience delayed general use of lipid emulsions (Alexander and Ziene 1961). Indeed, as late as 1973, the U.S. Food and Drug Administration had not approved the addition of fat preparations to parenteral formulas, and reports continued of essential fatty acid deficiency in infants (White et al. 1973). A 10 percent soybean oil emulsion, which employed a nontoxic emulsifying agent, egg phospholipid, and a smaller fat particle (0.5 microns in diameter), finally proved to be acceptable (Bivins et al. 1980).
Infants were not the only ones at risk for essential fatty acid deficiency from total parenteral nutrition. Adults with lesions of the gastrointestinal tract were reported to have biochemically defined linoleate deficiency (Wapnick, Norden, and Venturas 1974). Fourteen patients had triene–tetraene ratios with an average of greater than 2. Adults who had sufficient stores of adipose tissue fat were not believed to be at risk of essential fatty acid deficiency until the use of total parenteral nutrition (TPN) showed that this was not necessarily the case. When glucose and amino acids were infused continuously, lipolysis of adipose tissue fat was suppressed, and biochemical evidence of deficiency was present even if some fat was provided by oral nutrition in combination with parenteral feeding (Stein et al. 1980). By the mid–1970s, however, intravenous fat emulsions had become generally available, and it was accepted that a sufficient supply of linoleic acid was one of the important factors to be considered in every case of parenteral nutrition (Wolfram et al. 1978).
The Essentiality of Linolenic Acid
The earliest studies of unsaturated fatty acids (in common and chemical names; see Table IV.C.1.1) showed that both linoleic and linolenic acids had beneficial effects upon the clinical signs of deficiency in rats. Linoleic and arachidonic acids cured the deficiency’s symptoms of growth retardation, skin lesions, and excessive water consumption, whereas linolenic acid only cured growth retardation (Burr 1942). Attempts were subsequently made by a group of investigators at Berkeley to produce linolenic acid deficiency by maintaining rats for 3 generations on a diet lacking n-3 fatty acids. Levels of n-3 in tissues became very low, but small amounts remained and the rats showed no abnormality in growth, reproduction, or appearance (Tinoco et al. 1971). The Berkeley investigators then used radioactive carbon-labeled linolenic acid to trace the impact on dietary fat sources when linolenic acid was converted to docosahexaenoic acid (Poovaiah, Tinoco, and Lyman 1976). Measured in liver phospholipids, the radioactivity was recovered as 20:5n-3 and 22:6n-3. The dietary fat supplements containing n-6 fatty acids (linoleic and arachidonic acids) reduced the conversion of 20:4n-3 to 20:5n-3 (desatu-ration), whereas the n-3 supplements (18:3n-3 and 22:6n-3) reduced the conversion of 20:5n-3 to 22:5n-3 (elongation). The researchers concluded that 22:6n-3 may control its own formation by regulating elongation.
Table IV.C.1.1. Unsaturated fatty acids
|Chemical name||Common name|
|18:1n-9||9-octadecenoic||Oleic, 18:1 w 9|
|18:2n-6||9,12-octadecadienoic||Linoleic, 18:2 w 6|
|18:3n-6||6,9,12-octadecatrienoic||Gamma-linolenic, 18:3 w 6|
|18:3n-3||9,12,15-octadecatrienoic||Alpha-linolenic, 18:3 w 3|
20:3 w 6
|20:4n-6||5,8,11,14-eicosatetraenoic||Arachidonic, 20:4w 6|
EPA, 20:5 w 3
|22:4n-6||7,10,13,16-docosatetraenoic||Adrenic, 22:4 w 6|
|22:5n-6||4,7,10,13,16-docosapentaenoic||22:5 w 6|
DHA, 22:5 w 3
Evidence, though inconclusive, that linolenic acid is essential was gathered by examining tissues of rats depleted of the n-3 family. Brain and retinal tissue retained docosahexaenoic acid tenaciously through two generations of rat growth (Tinoco, Miljanich, and Medwadowski 1977; Tinoco et al. 1978), but prolonged deprivation of n-3 fatty acids resulted in reduced visual acuity in infant monkeys and defective electroretinographic responses in monkeys and rats (Neuringer, Anderson, and Connor 1988). As was the case with linoleic acid, patients requiring intravenous feeding have been important in proving that linolenic acid is essential in humans. Patients on TPN, observed by K. S. Bjerve (1989) from 1987 to 1989, experienced scaly and hemorrhagic dermatitis, hemorrhagic folliculitis of the scalp, growth retardation, and impaired wound healing. But the addition of different oils showed that 1.0 to 1.2 percent of energy as linolenate was necessary to obtain a normal concentration of n-3 fatty acids and relieve the symptoms.
The Discovery of Prostaglandins
Meanwhile, in Sweden, Sune Bergstrom and his colleagues (1962) had determined the structure of a new class of compounds that had been isolated from the vesicular glands of sheep and were named prostaglandins. The subsequent discovery that essential fatty acids were the natural precursors of the prostaglandins was made simultaneously by Bergstrom’s team, by a group in Holland, and by another at the Upjohn Company in the United States (Bergstrom 1972).That essential fatty acid deficiency could affect prostaglandin functions was shown by Bergstrom and L.A. Carlson (1965).
The first international conference on prostaglandins was held in 1972, and Bergstrom, who opened the meeting, commented on the difficulty of evaluating analytical methods for prostaglandins. At that time, bioassay and gas chromatography–mass spectrometry (GC-MS) were being used. Radioimmunoassay was quite new and considered unreliable, as were bioassays. Bergstrom (1973) called for intensive discussion of the analytical questions. Daniel H. Hwang, at the time a doctoral student in nutrition, proposed that in order to study dietary effects on prostaglandin status, a reliable radioimmunoassay should be developed. GCMS was not sensitive enough and was too time-consuming and expensive to be used for analysis of large numbers of biological samples.
Very little was known then about the metabolism of prostaglandins. The short half-life, explosive synthesis in response to trauma, and tissue specificity were yet to be discovered. Serendipitously, Hwang and colleagues (1975) chose to analyze blood serum from rats that had been anesthetized, and these experiments were the first to apply the discovery of prostaglandins to the understanding of essential fatty acid functions and requirements. Rats were fed diets containing corn oil or beef tallow as the source of fat, which showed that there was a positive effect of corn oil (containing linoleic acid) on the synthesis of PGE1 and PGF2α.
Subsequently, the laboratory of Melvin M. Mathias and Jacqueline Dupont (1985) demonstrated a biphasic response of prostaglandin synthesis to the dietary content of linoleic acid. The response to 0 to 2 percent of energy from linoleate was an increase in prostaglandin synthesis; with 2 to 5 percent of energy there was a decrease, and above 5 percent of energy there was a gradual increase up to the maximum content of linoleic acid fed (27 percent of energy).The 0 to 2 percent energy response to linoleate was associated with a disappearance of eicosatrienoic acid. Responses to a higher consumption of linoleate were not correlated with arachidonic acid concentration in serum.
The importance of the relation of dietary linoleate to eicosanoid synthesis meant the addition of a functional measurement to the earlier indications of essential fatty acid deficiency, both clinical and biochemical. This functional indicator of essential fatty acid requirement suggested that 5 to 10 percent of linoleate is desirable (Dupont and Dowd 1990).
Excessive extradermal water loss, one of the first symptoms described of essential fatty acid deficiency, has been explained. A major specific function of linoleate is in skin ceramides, where the linoleate is incorporated into acylglucosylceramides and acylceramides (Hansen and Jensen 1985). Linoleate is the only fatty acid substantially incorporated into these sphingolipids. On another front, the details of control of elongation and desaturation and interrelationships among the families of fatty acids are still being investigated (Cook et al. 1991; Cunnane et al.1995; Sprecher et al. 1995).
Since the 1970s, an enormous literature about prostaglandin metabolism has accumulated. The term “eicosanoids” was introduced in 1980 to describe the class of substances having 20 carbon atoms derived from n-6 and n-3 fatty acids. Because eicosanoids are regulators of a large array of physiological functions, there are many possible manifestations of deficiency of their precursors. The attention given to the very long-chain fatty acids from marine animals (fish oils) has created another large body of literature about the interplay between metabolism of omega-3 (n-3) and omega-6 (n-6) families of fatty acids. The competition between the two families for enzymes of chain elongation, desaturation, and conversion to active metabolites first demonstrated by Holman (1964) has introduced additional complexity into the attempt to define the dietary requirements for the two families. We know that a source of 18 carbon n-3 and n-6 fatty acids is a dietary necessity, but whether there may be health benefits from consumption of the longer chain products—20:4n-6, 22:5n-3, and 22:6n-3—is a current topic of intensive research. Infant requirements for brain development are important considerations for assuring appropriate recommendations for infant formulas and supplements (Carlson et al. 1993).
A broad range of ongoing research is aimed at defining the functions of n-3 fatty acids, and a deficiency has been associated with the function of rhodopsin (Bush et al. 1994). Many aspects of brain and behavioral development are linked to the availability of linolenic acid and its products (Neuringer, Reisbick, and Janowsky 1994).The effects of the ratio of dietary linoleic to linolenic acids is the subject of current research (Jensen et al. 1996), and immune functions and cell signaling are also current topics of study of fatty acid functions and metabolism (Hayek et al. 1997; Hwang in press).
The evolution of an understanding of essential fatty acids has progressed through several scientific developmental phases. The earliest was the ability to prepare diets of sufficient purity to exclude lipids, which led to the conclusion that some fat was essential. The substantive proof of a human requirement—that is, that the fatty acids were essential to the diet—had to await the advent of total parenteral feeding. In about the same era, eicosanoids were discovered, which opened a new world for understanding the functions of essential fatty acids. With the tools of science today, the profound participation of fatty acids in all aspects of cellular life and function should provide great excitement as well as great challenges to all scientists interested in the expansion of the science of nutrition.