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180.

ME TABOL ISH OF THE UNSA TURA TED AND SA TURA TED FA TS

F. A. KUMMEROW

No chemical distinction exists between plastic (shortening) and vegetable (salad oils); both contain connected to or "esterified" with three moles of fatty (Table 1). The saturated fatty in the comrnon edible fats vary from C4 to C22 in chain length and -7.9 to 79.9% in . The unsaturated fatty acids in edible fats vary from C to C20 in chain length and -1 to -49OC in melting point (Markley '60). '$fe high melting points of the saturated and the low melt- ing points of the unsaturated fatty acids are important to the physical characteristics of fats and oils.

If the glycerol is esterified with more than two unsaturated fatty acids, i.e., oleic or , the resulting is a liquid or an ''" at room . If, on the other hand, glycerol is esterified with only long chain saturated fatty acids or only one mole of oleic and two moles of palmitic or , the resulting triglycer- ides is a solid or "" at room temperature (Table,2). A study of isolated has shown that the substitution of one mle of oleic for stearic acid in an d -position in tristearin for example lowers the melting point from 73 to 38OC and the same substitution in from 66 to When one considers that the body temperature of a 35OC (Bailey '50). human being is 37.ZoC, the & substitution of linoleic or for one mole of stearic or may change the physical character of the depot fat and its ability to act as EL "cushioning agent" to vital organs. These depot fats may become soft and oily.

Natural fats and oils have been found to contain mixtures of triglycerides which are uniquely characteristic of a specific fat (Hilditch '56). As indicated by the melting points of isolated triglycerides, the physical properties of the mixture of triglycerides are governed by the physical properties of the particular which is esterified with the glycerol (Table 3). In ''soft" fats such as corn or , which contab the unsaturated oleic and linoleic acids as the predominant fatty acids, "the oils" are composed of a high proportion of di and tri unsaturated . In "hard" fats such as oil, and beef , which contain the saturated, myristic, palmitic and stearic acids as the predominant fatty acids, "the fats" are composed of a high proportion of di and tri saturated glycerides. Although is a , it is classified as a fat as it contains 84% tri saturated glycerides and is a solid at room temperature. Human fat and human milk fat are semi solid fats with a high proportion of mono and di saturated glycerides. 181.

The glycerides of human adipose tissue contain approximately 4% myristic, 25% palmitic, 7% stearic, 6% palmitoleic, 46$ oleic and 2% of fatty acids which are shorter than 14 or longer than 18 atoms in chain length (Cramer and Brown '43). These fatty acids may be classified the "non essential fatty acids" as they can all be synthesized in the body from non fat precursors. They are also found in , beef tallow and but in different percentage composition in each case. In addition the adipose tissue fat is composed of approximately 9% linoleic and 1% which contain two and four double bonds respectively. These two fatty acids have been classified the "essential fatty acids" as linoleic acid cannot be synthesized by animal tissue and serves as an essential precursor for the synthesis of arachidonic acid.

The ability of unsaturated fatty acids to form "geometric isomers", plays an important role in the degree of hardness of a "hydrogen- ated" fat such as (Table 4) and probably of human depot fats. Geometric isomers are important to the plasticity of fats because the -trans isomers of oleic and linoleic acid, which are produced during the commercial of an edible oil have melting points of 52OC and 29OC respectively and therefore tend to "harden" margarine. The natural cis isomers of oleic and linoleic acid have melting points of 14OC and mCrespectively and therefore tend to "soften" margarine at room temper- ature or 21OC. This property of oleic and linoleic acids to exist in either a solid or a liquid state at room temperature is important to the produc- tion of of high linoleic acid content for two reasons. One, even though the trans isomers of oleic and linoleic acids influence the plasticity of margarine, these trans isomers have the same degree of un- saturation or number as the natural cis isomers and therefore both contribute to the calculated tfpolyunsaturates" and both thus increase the polyunsaturated to saturated or P/S ratio of the fat. Two, the deliberate production of the high melting trans oleic and linoleic acid during com- mercial hydrogenation allows the margarine mnaufacturer to add a higher percentage of the low melting linoleic acid to a margarine fat without sacrificing the degree of desirable hardness. Thus mdern margarines, whether made from corn oil, cottonseed oil, or , all contain two to three times more linoleic acid then a few years ago, but they also con- tain more trans oleic acid.

We purchased four typical brands of both high and low priced margarine at a local supermarket and subjected them to fatty acid and infrared analysis (Table 5). The results (Kummerow '64) indicated that all of them contained more linoleic acid than the margarines which were avail- able a few years ago (Bailey '51). Furthemre, the cost of these margarines was independent of their linoleic acid content; the lowest priced margarine contained 15% more linoleic acid than the medium priced brand and only 54 less linoleic acid than the highest priced brand. The large arnount of "trans" fatty acid in all of the margarines indicated that both trans oleic and trans, trans linoleic acid may have been produced during the hydrogen- ation of soybean oil, which forms the stock of most margarines.

The trans fatty acids present in human tissue apparently arise solely from dietary fat, and as in rats, they do not normally appear in the tissues unless a source of trans fatty acids is included in the diet. Samples of fat from human placental, maternal., fetal, and baby tissue were 182. examined for the presence of trans fatty acids. While the maternal tissue contained considerable amounts of trans fatty acids, these were not found to any measurable extent in placental, fetal, or baby fat (Johnston '58) a

The percentage of trans fatty acids in rat fat decreased when trans fatty acids were removed from the diet (Johnston '58a). However, they did not completely disappear from the tissue even at the end of two months on a diet free of trans fatty acid. After one month on the diet free of trans fatty acid, the carcass fat of the rats which had received 10% of margarine stock had decreased from 18.6 to 6.5% and after two months to 4.4% of trans fatty acids. The carcass fat of the animals which had re- ceived margarine stock and contained approximately 11% of trans fatty acids. After one month on a diet free of trans fatty acids, the car- cass fat decreased to 4.9% and after two months to 2.8% of -trans fatty acids. It seems evident that the high trans fatty acid content of marga- rine fat could "harden" human depot fat and counteract the "softening" in- fluence of linoleic acid. The of such depot fat would indicate a higher P/S ratio. However, the melting point and other physical characteristics of the depot fat might not be changed significantly from a depot fat which contained stearic instead of trans oleic acid, that is someone eating butterfat instead of margarine.

The palmitic and stearic acid which is found in tissue fat does not have to be consumed as a component of dietary fats. It has been shown with the aid of CI4 labeled (two carbon atoms long in chain length) that fatty acids can be shortened or elongated --in vivo so that triglycerides specific to each species can be synthesized in animal tissue. For example (Table 6), it has been shown that stearic acid can be converted to palmitic acid through the collaboration of five different and the presence of the proper cofactors (Bloch '60). In the overall reaction adds to stearic acid and two carbon atoms are removed as acetyl Co A. The resulting palmityl Co A can add --in vivo to a dig1ycerj.de to pro- duce a triglyceride which contains one mole of pdmitic instead of stearic acid. When it is not needed for triglyceride synthesis, the palmityl Co A can be degraded until all of it is converted to acetyl Co A.

The acetyl Co A, in the presence of bicarbonate, and , can be carboxylated to form malonyl Co A (Table 7). The malonyl Co A in the presence of reduced triphosphopyridine nucleotide (TFNH) and with the elimination of water can be converted back to palmitic acid (Lynen '61). In the process of synthesis, both palmitic and stearic acid can be dehydrogenated to palmitoleic or oleic acid respec- tively. Thus with the aid of a dietary source of essential fatty acids, animal tissue can produce fatty acids of proper chain length and the degree of unsaturation which is best suited for its needs. However, the excessive consumption of dietary sources of essential fatty acids such as corn oil will contribute to the "metabolic pool" of acetyl Co A as effectively as an excessive consumption of animal fats. Furthemre, when tissues are flooded with large arrounts of a highly unsaturated fat, they appear to accumulate in tissues in abnormal amounts (Chu and Kummerow '50). (Table 8). Under normal conditions carbohydrates furnish the major raw material for the synthesis of fatty acids. (Table 9) by means 183.

of oxidative decarboxylation forms acetyl Co A. Metabolic pathways are also available for the synthesis of fatty acids from amino acids. The glucogenic amino acids are convertible to pyruvic acid; the ketogenic amino acids form or acetoacetate both of which are lipogenic. In all cases acetyl Co A is the immediate starting material for the formation of fatty acids.

The "metabolic pool" of acetyl Co A does not exist as such but is in a continuous state of . If the dietary intake of metabolites is just sufficient or is made deficient by the excessive use of muscles and acetyl Co A is used up in the cycle to produce heat and energy (I), the conversion of acetyl Co A to fatty acids (11) and (111) would be minimal. However, if the total caloric intake is in excess of energy and maintenance requirements, acetyl Co A is converted to fatty acids and cholesterol. The major portion of the excess serum cholesterol is convertedto bile acids in the and excreted. However, the excess fatty acids are deposited as triglycerides and along with cholesterol, and other lipids add to the unwanted deposits of tissue fats. It is therefore essential to balance the energy requirements against total caloric need in order to prevent an accumulation of tissue fats. The adipose tissue fat and serum cholesterol level can be reduced by increasing energy expenditures or by decreasing caloric intake. However, the obesity problem attests to the fact that it is difficult to carry out 812 orderly of in an atmosphere of dietary abundance.

The highly unsaturated fatty acids have been divided into three families (Mead '60), the oleic, linoleic, and families re- spectively (Table 10). Curing their metabolism these fatty acids are elongated and desaturated to a series in which the first double bond is located at the 9th, 6th or 3rd position from the methyl end of the fatty acid chain. The elongated oleic family is characterized by the CH3(CH2)3 ending; it exists to an appreciable extent in fat-deficient animals. In such animals a considerable amount of a C20 triple unsaturated 5,8,11- eicosatrienoic acid is formed by elongation of oleic acid, by the addition of acetyl Co A and by desaturation of the carbon chain. The linoleic de- rived family present in dietary fats is characterized by the CH3(CH2)4 terminal group of the "essential" linoleic acid and its elongated deriva- tive, the C20 arachidonic acid. The linolenic family is characterized by the CH3CH2 end group and is found in the serum lipids of animals fed linolenic acid. Holman and Mohrhauer ('63) believe that when linolenic acid is present in the dietary fat its conversion to higher unsaturated fatty acids takes precedence over the metabolism of linoleate by a factor near tenfold. Linoleate metabolism proceeds in preference to oleate metabolism and oleate metabolism to higher unsaturated acids can take place only when linoleate and linolenate are present in low concentration.

Mead ('60) has traced the steps involved in the conversion of linoleic to arachidonic acid. However, to date, the degradation of linoleic acid has not been fully elucidated. It is not known whether un- saturated fatty acids are first biohydrogenated and then degraded into two carbon units or whether they are desdurated further before they are metabolized. We are presently following the metabolism of tritium labeled linoleic acid, which has been prepared in our laboratory, and hope to clarify this point in the near future. 184.

An interesting relationship between the three families of unsatu- rated fatty acids (Table 11) has been noted when they are incorporated into deficient diets. All three families of unsaturated fatty acids cause exudative diathesis in chick and muscular dystrophy in rats, rabbits, sheep and cattle. However, only the essential fatty acids of the linoleic acids series cause chick encephalomalacia (Kummerow '64).

Since polyunsaturated fatty acids are incorporated into the lipids which are involved in the surface structure of the wall, dietary fac- tors may exert some influence on the integrity of the cells. For example (Walker '64) variation of the dietary fat and the omission of Vitamin E fromthe diet resulted in changes in the stability of erythrocytes. Vitamin E deficiency resulted in the most significant changes, whereas the nature of the dietary fat tended to modify the degree of change. The cells from Vitamin E-supplemented rats showed little or no hemolysis; with corn oil the degree of hemolysis was greater than with the more saturated lard. Re- placement of cellular oxygen with carbon monoxide inhibited this hemolytic activity, which is consequently believed to be oxidative in nature.

In another series of experinents, the importance of the essential fatty acids to the structural integrity of the cell was studied (Walker '64). An increasing amount of dietary linoleic acid as supplied by coconut oil, butterfat, and corn oil resulted in increased incorporation of linoleic acid into the of erythrocytes and also to increased arachidonic acid incorporation (Table 12). Where dietary linoleate was re- stricted, more palmitoleic and oleic acids were incorporated into the cellular lipids, and the eicosatrienoic acids characteristic of deficiency were also found in increasing amounts, comprising over 16% of the total fatty acids when hydrogenated coconut oil was the dietary fat.

The erythrocytes fromthese animals were subjected to hemolysis by isotonic solutions of three non-electrolytes glycerol, thiourea and tri- . With each solute studied, the hemolysis resulting from the permeation of the solute into the cell was most rapid in cells from the animals fed coconut oil. As the dietary linoleic acid intake increased, the rate of hemolysis decreased. It is possible that hemolysis reflected structural changes arising in the erythrocyte membrane from the incorpora- tion of specific fatty acids.

In a recent report, Vendenheuvel ('63) advanced a model for bio- logical organization at the molecular level. This model involved a complex resulting from the association of cholesterol with or glycerophosphatide and was applied specifically to the structure of the myelin sheath. It is interesting, however, to consider the possibility of the role of the essential fatty acids in the of such complexes (Fig. 1). In a representation of constructed geometrically from the parameters given by Vandenheuvel, the d -position of the glycerol moiety is esterified with stearic acid (ABC) and the &-position with arachidonic (ABDE) or 5,8,11-eicosatrienoic acid (ABDF). In a complex such as that proposed by Vandenheuvel, the curvature of the arachidonic acid chain would result in greater stearic hindrance to the cholesterol than would mono- or dienoic acids. However, the substitution of the trienoic acid for the arachidonic acid also results in an increase in the over-all width of the 185. lecithin moiety. This increase, Y, is about 20% of the width, X, of the arachidonyl-lecithin. It is tempting to speculate that some of the prop- erties of cell membranes may be governed by the type of fatty acid in the complex. For example, when the oleic or linolenic series of polyunsatu- rated fatty acids are incorporated into the cell at the expense of arachidonic acid, a change in structure of the molecules may occur and may result in a looser packing of the phospholipid complexes in the membrane thus altering its stability and permeability.

It is interesting to note that the c18 -cis-9, trans-12, octadecadienoic acid, a possible component of hydrogenated soybean oil, can be elongated and desaturated to the Czo, 5,8,11,14-, the C18 trans fatty acid will not prevent the symptoms of essential fatty acid deficiency. The Cz0 fatty acid is a geometric isomer of arachidonic acid with atrans double bond in the 14- position. The orthogonal projec- tion of a phosphatide containing this C20 fatty acid would be very similar to that of the phosphatide containing the non-essential eicosatrienoic derived from oleic acid and may also alter the stability and permeability of erythrocytes. Thus a simple change in the composition of the unsaturated fatty acids in the tissue lipids may influence the integrity of cell membranes.

SUMMARY

In summary, dietary fats represent the most compact energy source available to man. However, dietary fats should not be thought of solely 88 providers of unwanted calories as fats are as vital to cell structure and biological function as protein. Tissue fat can be synthe- sized from either carbohydrate or protein, therefore, the total caloric intake rather than any one dietary component is crucial to the amount of deposition of lipids into the tissue.

An optimum intake of essential fatty acids may be important to the integrity of the cell wall of erythrocytes. However, until the entire picture of the role of dietary fats in optimum nutrition is clarified, it would seem judicious to consume a well-balanced diet of , milk, eggs, vegetables, fruits, and sufficient cereals and bread to provide for an ade- quate protein, vitamin, and caloric intake. The optimum total intake of linoleic acid by man has not been established. The level of linoleic acid in the American dietary pattern could be increased through the availability of less severely hydrogenated shortenings but the indiscriminate dietary substitution of "soft" for "hard" fats seems undesirable.

REFERENCES Bailey, A. E. 1950 Melting and Solidification of Fats. Interscience Publishers, New York, p. 166.

Bailey, A. E. 1951 Industrial Oil and Fat Products. Interscience Publishers, New York, p. 759. 186.

Bloch, K. 1960 Metabolism. John Wiley & Sons, New York, p. 41. Chu, T. K. and F. A. Kummerow 1950 The Deposition of Linolenic Acid in Chickens Fed . Poultry Sci., -24: 846.

Cramer, D. L. and J. B. Brown 1943 The Component Fatty Acids of Human Depot Fat. J. Biol. Chem., 151: 427. Hilditch, T. P. 1956 The Chemical Constitution of Natural Fats. John Wiley & Sons, New York, p. 391.

Johnston, P. V., D. C. Johnson and F. A. Kummerow 1958a Deposition in Tissues and Fecal Excretion of Trans Fatty Acids in the Rat. J. Nutrition, -65: 13. Johnston, P. V., F. A. Kummerow and C. H. Walton 1958 Origin of Trans Fatty Acids in Human Tissue. Doc. SOC. Exptl. Biol. Med., -99: 735.

Kummerow, F. A. 1964 The Possible Role of Vitamin E in Unsaturated . Fed. Proc., in press.

Kummerow, F. A. 1964 The Role of Polyunsaturated Fatty Acids in Nutrition. Food Tech., in press.

Lynen, F. 1961 of Saturated Fatty Acids. Fed. Proc., g:941.

Markley, K. S. 1960 Fatty Acids. Interscience Publishers, New York, p. 34. Mead, J. F. 1960 Metabolism of the Polyunsaturated Fatty Acids. Am. J. Clin. Nutrition, -8: 55. Mohrhauer, H. and R. T. Holman 1963 The Effect of Dietary Essential Fatty Acids Upon Composition of Polyunsaturated Fatty Acids in Depot Fat and Erythrocytes of the Rat. J. Lipid Res., -4: 346.

Vendenheuvel, F. A. 1963 Study of Biological Structure at the Molecular Level with Stereomodel Projections. I. The Lipids in the Myelin Sheath of Nerve. J. Am. Oil Chem. SOC., -40: 455. Walker, B. and F. A. Kummerow 1964 Dietary Fat and the Structure and Properties of Rat Erythrocytes. J. Nutrition, -82: 323. Walker, B. and F. A. Kummerow 1964 Erythrocyte Fatty Acids and Apparent Permeability to Non Electrolytes. hoc. SOC. Exptl. Biol. Med., -115: 1099. 187.

TABLE 1

Melting; Points of Fatty Acids

Saturated

m.p .OC m.p .OC c4 Butyric -7.9 C14 mistic 54.4

c6 Caproic -3.4 c16 Palmitic 62.9

cg Caprylic 16.7 C18 Stearic 69.6

Cl0 Capric 31.6 Cz0 Arachidic 75.3 C12 Lauric 44.2 C22 Behenic 79.9

Uns aturated

'16 :1 Palmitoleic -1 %8:1 Oleic 14

Linoleic -12 %8:2

c20:4 Ar ac h idonic -49

TABm 2 The- Effect of Unsaturated Fatty ---Acids on the MeltiG ----Triglyceride

Glyceride -M.P. -M.P. s-s-s 73% P-P-P 66OC

s-s-0 38 P-0-P 35 s-0-s 43 P-0-0 19

s-0-0 23 M-0 -0 14

L-0-0 7

O-Oleic; P-Palmitic; S-Stearic Acid; L-Linoleic; M-Myristic 188.

TABLE 3 Glyceride Composition -of Vegetable -and Animal Fats-

GS3 GSzU GSU2 A- -?b 2 corn oil 1 15 45 38

Cottonseed oil 0 13 44 43

Coconut oil 84 12 4 0

Butterfat 35 36 29 0

Beef tallow 15 46 37 2

Lard 2 26 55 17

Human (adipose) 5 26 43 24

Human (milk) 9 40 43 8

S - Saturated fatty acid U - Unsaturated fatty acid

TABLE 4 Melting Points ----of Cis and Trans Isomers

C------x ll x-----c11 C Y C Y Oleic Elaidic m.p. 14'C m.p. 52OC

Xd11 X4 I1 C+4 C----c-----c II ll C Y C Y

Linoleic Linoelaidic m.p. -12'~ m.p. 29OC 189.

TABLE 5

Comparative Composition of Margarines

Fatty Acid M39# F39# A29k Palmitic 14.7 16.2 9.4 11.7

Stearic 7.3 9.3 5.1 13.0

Totd sat. 22.3 25.7 14.8 25 .O

Oleic 41.7 43.4 73.3 47.5

Linoleic 35.4 30.2 10.8 25.8

Total Unsat. 77.1 73.6 84.1 73.3

Total "Trans" 28.7 19.6 43.6 48.6

TABI;E 6

Conversion of Stearic Acid to Metabolic Products

a3(CH2)16CmH + 2HSCoA (Stearic Acid) + (Coenzyme A)

CH3(CH2) 14COOH + CH3COSCoA (Palmityl CoA) + (Acetyl CoA) 1 BCH~COSCOA(Acetyl CoA) 190.

TABLE 7 The- Synthesis -of Palmitic Acid

CH3COSCoA + HCO3 +HOOCCH2COSCoA (Acetyl CoA) (Carbonate) (Malonyl CoA)

CH~CHZCH~COSCOA(Butyryl CoA) J 1 CH; ( CH2) 14COSCoA (Palmityl CoA) &

TABLE 8 Acetone Soluble -and Insoluble Oleic

-and Linolenic ---in Skin Fat

Dietary Oleic Linolenic Lin. oil Soluble Insoluble Soluble Insoluble

0% 23.3% 30.8% 0.8% 0.3%

6% 16.2% 39.$ 20.7% 0.9%

1% 24.7% 47 .s$ 22.276 1.1%

2570 21-85 50 .l$ 28.0% 1.2% 191.

TABLE 9 Relationship -of Metabolites

Carbohydrate Protein 4 Pyruvate -C02 Acetyl -m2 amino4 +&A ' +CoA acids

A I-I I1 I11 I I Citrate Oxbo - A Long'chain steio 1s acetate fatty acids ( CH~-CHZ)~COOH u coz+H20 -2H bile Oleic acid acids + depo sited excreted1 in tissue (heat & energy)

TABLE 10 Metabolites of the Three Unsaturated Fatty Acid Families

All 9 eicosa- eicos a- oleic -b dienoic _____.) trienoic 18:l 20:2 20:3

All 6 machi- doc0 6 a- linoleake donate pentaenoic 18:2 .-b 20:4 -b 22:s

All 3 eicos a- doc 0 S a- linolenate -+ pentaenoic .-) pentaenoic 18:3 20:5 22:s

6,9,12,15 arachidonic acid 192.

TABLE 11 Pathology Caused bz Vitamin E, Deficiency

Patho logy symptom Case Delayed by 1. Exudative diathesis Edema PUFA Se

2. Myopathy rnscular S amino dystrophy 11 acids

3. Encephalo- Spasm or EFA Linolenic malacia paralysis series

TABLE 12 Fatty Acid Composition of the Erythrocyte Lipids

Acid- Coconut 0i 1 Castor oil corn Oil 16:O 22.4$ 2s. 2% 24.2$

16:l 2.7 1.7 0.4

18:O 14.2 13.5 13.5

18:l 15.7 13.5 8.6

1a:2 2.2 5.3 11.5

20:3 15 .O 1.3 0.1

20: 3 1 .o 1.1 0.5

20:4 15.4 26.3 31 .O 153. PHOSPHATIDYL

C

0 GLYCCEROL Cm

0 OXYGEN -0-

OXYGEN =O

. .e...... @ .. OXYGIEN -0- V .....d .... .’ F NITROGEN

A PHOSPHORUS ABC - STEARIC ACID

ABDE - ARACHIDONIC ACID ABOF - ECOSATRIENOIC ACID

Figure 1

DR. KASTEWC: Thank you, Dr. -row. I thipk we will withhold questioning until after we have had an opportunity to hear the next paper. I take great pleasure in introducing Dr. Hector DeLuca, who is a member of the Department of here at the University of Wisconsin, a well known biochedst. I understand now that he is following the interest of Dr. Steenbock, as might be surmised fromthe topic of his discussion this after- noon. I take pleasure in welcoming him to this group. Dr. DeLuca,

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