ROBERT L. ANDERSON, CURTIS S. FULLMER, JR.2 AND EDWARD J. HOLLENBACH The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45247 ABSTRACT Weanling rats were fed diets that contained 2.5% linoleate (cc-18:2) along with graded levels (1.2-18%) of its irons isomers, either the di-trans isomer (tt-18:2) or a mixture of the mono-frans isomers (ct,tc-18:2). The trans isomers were added at the expense of oleate. After a 6-week feeding experiment produced no signifi cant differences in weight or feed efficiency among the rats, the fatty acid compositions of their depot fats and liver lipids were determined. The depot fats closely resembled the dietary fats, with tt-18:2, tc-18:2, and ct-18:2 being deposited at levels proportional to their levels in the diets. The liver lipids showed a preferential accumulation of cc-18:2, and although tt-18:2 and tc-18:2 were deposited at levels proportional to their dietary concentrations, ct-18:2 did not appear in the liver lipids to an appreciable extent. Apparently it is excluded from the phospholipids which comprised 65% of the liver lipid. Ratios of 18:2 to 20:4 in the liver were also studied. Increasing levels of dietary tt-18:2 caused decreased levels of arachidonate in the liver, suggesting that chain elongation of cc-18:2 is somewhat inhibited by high ratios of tt-18:2 to cc-18:2. This inhibition was appreciable only at tt-18:2/cc-18:2 ratios greatly in excess of the ratios that occur in human diets. The presence of ct,tc-18:2 in the diet did not affect the level of arachidonate in the liver, but did give rise to a rrarw-20:4 fatty acid, presumably by chain elongation of ct-18:2. Although tc-18:2 serves as an energy source, it is neither a substrate nor an inhibitor for chain elongation. J. Nutr. 105: 393-400, 1975. INDEXING donic acid
KEY WORDS
linoleic acid metabolism
The geometrical isomers of unsaturated fatty acids occur widely. Because they are formed by the partial hydrogénation of polyunsaturated fats in the rumens of sheep and cattle, they appear in the milk and body fats of these animals. Because they are formed by the partial hydrogénationof polyunsaturated fats, they appear in commereiai shortenings and margarines. Because of their evident importance, a substantial body of information has been built up about the digestion, absorption, and catabolism
of both
trans-l8:l
and
ienmpr« nf linnlpiri IK .«i 01 tne trans isomers or nnoieic
acid
(18:2) on the conversion , i, . ' . j , nr. .* , arachldoniC acid (20:4) have
OÕ 18:2 tO , , j. j been Studied
in both the intact animal (1-6) and at the enzyme level (7). In the intact animal the io n • 18:2 ISOmer
-it With
two
trans
i ui double
•arachi-
inhibits 20:4 synthesis (1-5), but the 18:2 isomers with one trans bond do not in fluence 20:4 synthesis ( 1 ). It has also been shown that the 18:2 isomer with two trans bond inhibits the microsomal enzyme system that converts linoleic acid to y-linolenic acid (7). It has also been demonstrated that ct-18:2 but not tc-18:2 can be converted to a 20:4 with a trans double bond (5). There exists some controversy as to •
tnns-
io n r u • J lo:Z tatty aCIUS. Tbp pflW'ts nf tVip franc
•trans fatty acids
Receivedfor publication June 20.1974. Abbreviations : in the designation of the geometric Isomers of linoleic acid, the first letter refers to the configuration of the double bond at the 9,10-posltion
nnd the gecondletter refers to the configurationof the double bond at the 12.13-posltlon ; c = ci»; t = trans. For example. ct-18:2 = 9-CÃ-»,12-Ã-rThe ct,tc-18:2 used to prepare these diets had a ct/tc ratio of 0.6.
whether tt-18:2 can be converted to a 20:4 acid with two trans bonds. Knipprath and Mead (8) have presented evidence that tt-18:2 is elongated to 20:4, while Privett et al. (5) and Kimura and Tsuchiya (2) observed no 20:4 with irons bonds when tt-18:2wasfed. In the work reported here, the 18:2 isomers with one or two trans bonds were fed to rats at graded levels with a constant supply of cc-18:2 to ascertain the level of trans-18:2 required to influence the con version of cc-18:2 to 20:4. The report also describes the liver and adipose tissue con centrations of the 18:2 isomers as a func tion of dietary level. TABLE 2 Composition of the diets
Casein Sucrose Water-soluble vitamins in sucrose1 Salt mixture U.S.P. XIV2 Cellulose3
Fat-soluble vitamin mixture4
Fat
18
M 5 4
:< l
19
100 1 The water-soluble vitamin mixture containined (in mg) : menadiune, 0.3: thiamin-HCl, 0.4; riboflavin, 0.7: niacin, 2.0; calcium pantothenate, 0.2; folie acid, 0.002; pyridoxine, 0.4; vitamin B-12, 0.015; choline chloride, 300; inositol, 200; ascorbic acid, 10 ; biotin, 0,015 ; p-aminobenzoic acid, 10. 1 Nutritional Biochemicals, Inc., Cleveland, Ohio (23). * Celluflour, Chicago Dietetic Supply House, Chicago, 111. ' The fat-soluble vitamin mixture was prepared using the appropriate fat for the diet. It contained (in U.S.P. units) : retinyl acetate, 1,250; ergocaleiferol, (1,250); o-a-tocopherol, 10.
MATERIALS AND METHODS
Preparation of fats. Pure methyl linoleate was obtained by low temperature urea ad duction of safflower oil methyl esters. Treatment with SOL>converted it to a mix ture of geometrical isomers (9). Low tem perature crystallization yielded two frac tions; one was the isomer with two trans bonds (tt-18:2), and the other was a mix ture of the isomers with one trans bond ( ct,tc-18:2 ). Analysis by argentation thinlayer chromatography (4) showed the presence of a small amount of mono-irons material as a contaminant in the tt-18:2 preparation, while the ct,tc-18:2 prepara tion contained only traces of the di-trans isomer. The methyl esters were transesterified with glycerol and alkaline catalyst to con vert them to triglycérides,each containing a single fatty acid. These were mixed with other pure triglycéridesin chosen propor tions, and the mixtures were randomized by treatment with sodium methoxide under nitrogen. The resulting triglycéridesconsti tuted a series having trans-18:2/cc-18:2 rations of 0.5, 1, 2, 4, and 8, with each product containing 2.5% of cc-18:2. Thus, as shown in table 1, several levels of tt-18:2 or of ct,tc-18:2 were added to the diet at the expense of 18:1. The triglyc éridesthat contained ct,tc-18:2 were made from two different preparations of this isomer mixture, and they differed in their ct/tc ratios as shown in table 1.
3(J5
METABOLIC EFFECTS OF iraus-LINOLEATE 5,8,11,14-20:4
5,8,11-20:3 8,11,14-20:3
B.
trans-20:4?
80
82 Time(min.)
84
86
Fig. 1 Tracings of Cao-polyenoic acid region of capillary chromatographs of liver lipid fatty acid methyl esters from animals fed (A) control diet; (B) tt-18:2, diet E (table 1); and (C) ct,tc-18:2, diet E (table 1).
lary GLC column. Retention times of their components were then compared with re tention times that had been established for some specific structures. Knowledge of double bond positions came (13) by application of Downing and Greene's cleavage technique. The trans bond content of the isolated 20:4 was determined by infrared spectroscopy (14). One analytical ambiguity remained. The capillary GLC column did not separate tc from tt-18:2, and therefore the composi tion of this peak in the samples of animal lipid could not be established by GLC evidence alone. But it was known from argentation TLC that the di-trans product contained only a small amount of monotrans material, and that the mono-trans mixture contained very little di-trans con taminant. Because the materials being fed were thus clearly defined, and because only peaks with the same retention time as those in the dietary fat fed were observed, we re3Partition liquid and solid support were obtained from Applied Science Laboratories, State College, Pa. « Hewlett-Packard model 720.
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Feeding experiment. The triglycérides were incorporated into semisynthetic diets at a level of 20% (table 2). The diets were fed to weanling, male, Sprague-Dawley rats (five rats per diet) for 6 weeks. The rats were housed individually in cages with raised wire floors. The animals were housed in a room with constant temperature (25°) and humidity (52% of saturation) and a 12 hour light-dark cycle. Fresh feed was provided twice per week, and body weights were determined each week. The animals were killed after consuming the diets for 6 weeks, and their livers and epididymal fat pads were excised, rapidly weighed, frozen, and stored at —20° until analyzed. Total lipid extracts of the individual livers and of the pooled fat pads from each di etary group were prepared (10). Methyl esters of the fatty acids were prepared by refluxing the total lipid extracts with 2% H^SO4 in methanol in a nitrogen atmo sphere. The methyl esters were freed of cholesterol by chromatography on Florisil (11). Fatty acid analyses. The fatty acid com positions of the lipid materials were deter mined by gas-liquid chromatography (GLC) of their methyl esters. Separations were made on a 10 foot X 1/4 inch stain less steel column packed with 15% EGSS-X on 60/80 mesh Gas-Chrom P.3 Thermalconductivity gas chromatography was used with the following operating conditions: column oven temperature 190°,injection port 330°, detector 350°,flow rate 79.5 ml/minute.4 The 18:2 isomers and the C2o-polyenoic acid isomers were analyzed on a polyphenyl ether capillary column as described by Kuemmel and Chapman (12). The column was held at 180°,and the inlet pressure was increased from 30 to 60 psi at 1 hour ( after elution of the 18:2 isomers) to sharpen resolution of the C20 peaks. On this column it was possible to separate the 18:2 esters into cc-18:2, ct-18:2, and a mixture of tc-18:2 and tt-18:2. Figure 1 shows some separations of the C20-fatty acid esters on the capillary col umn. As indicated on the graph, some of the individual components were identified. This was done by collecting the 20:3 and 20:4 esters from the packed GLC column and rechromatographing them on the capil-
390
ANDERSON,
FULLMER
AND HOLLENBACH
TABLE 3 Amount and phoapholipid content of lipid in the livers of rats fed trans isomers of linoleic acid Diet
Weight
Epididymal fat pad1
Controltt-18:2 diet Adiet Bdiet Cdiet Ddiet Ect,tc-18:2 diet Adiet Bdiet Cdiet Ddiet Ea252
Liver lipids
a'4.2±0.2°4.0±0.1°4.1±0.1«3.8 100 phospholipid*63
±1°66 ±10«278±10°251 ±0.1«2.1 ±1°66±2°64±1«64±1«63±1 8"251 ± ±0.1«1.8±0.2°2.0 7°243 ± ±0.3°4.5 4°262 ± ±0.1«2.1 ±0.3«5.3 ±0.2«1.8±0.2°2.3±0.1°2.2±0.1°2.1 ±0.3»4.6±0.2«4.5 7°247 ± 5«251 ± ±10°248 ±0.2°4.5±0.5°4. ±1°65 ±12"257 ±1°64±1°63 5 ±10°255 ±0.1«2.2±0.1«gl±0.4"4.6±0.2°% ±1« ± 4«al
1 Tissue weight g/100 g body weight. Mean ±-IM of five animals. ' Grains of lipid per 100 g of liver. Mean ±SEMfor five animala per treatment ; numbers with same letter superscript are not significantly different from each other. * Percent phospho lipid determined by chromatography on acid-washed Fiorisi! (24).
ported the equivocal peak as tc-18:2 when it occurred in the lipids of animals fed ct,tc-18:2, and as tt-18:2 when it occurred in the lipids of animals fed tt-18:2. Statistical analyses employed analyses of variance as outlined by Snedecor (15).
affected the fraction of phospholipid in the liver lipids. Feeding the trans isomers to rats influ enced the compositions of their liver and depot lipids. The following changes were produced by the tt-18:2.
RESULTS None of the 11 diets produced any sig nificant differences in body or epididymal fat pads weights. As shown in table 3, the liver lipid content was slightly, but not significantly, elevated in all of the groups fed ct,tc-18:2. Among animals receiving tt-18:2, the liver lipid content was not sig nificantly elevated above the control level except in the animals fed the highest level of tt-18:2. None of the dietary treatments
1. In the epididymal fat pads, the frac tion of tt-18:2 increased with increasing levels of tt-18:2 in the diet, while the frac tion of cc-18:2 stayed constant (fig. 2A). 2. In the liver lipids, the fraction of tt18:2 increased with increasing levels of tt-18:2 in the diet. The fraction of cc-18:2 also increased up to the point where the diet contained 9% tt-18:2 and then re mained constant (fig. 3A). Even so, the
All t_u 20 4
».II.U-20,»
0
5
10 15 20 % 11-18:2in tht Ditl
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g2.0±0.2°2.1 100
5 O 15 20 let,te -18:2 in the Di«
Fig. 2 The effects of dietary tt-18:2 (A) and ct,tc-18:2 (B) on the 18:2 geometric isomer con centrations in epididymal fat pad fatty acids. Obtained on epididymal fat pads pooled from five animals per treatment.
% 11-18:2 i" Ihr D«!
Fig. 3 The effects of dietary tt-18:2 on the concentrations of the 18:2 isomers (A), and G»polyenoic acids (B) in rat liver fatty acids. Each point represents the mean ±standard error of the mean for five animals.
METABOLIC
EFFECTS
TABLE 4 Ratios of ct-18:e lo tc-18:2 in the diets, livers, and adipose tissues of rats fed diets containing various levels of these isomers Diet
Liver
Adipose tissue
ct-lS-.Z/tc-lS'.Z10.34
1.21'0.613Ratio ±0.07(10) 0.17±0.01(15)1.282
0.613
1 Mean ±SEM;numl>er of samples indicated in parentheses. ! Animals fed oils ct,tc-18:2, diets A and U (see table 1). • Animals fed oils ct,tc-18:2, diets C, D, and E (table 1).
3. The fraction of arachidonate in the liver lipids was not affected by additions of ct,tc-18:2 to the diet (fig. 4B). The dietary ct,tc-18:2 did give rise to increasing levels of a fatty acid that was identified (by capillary GLC retention time and IR) as a 20:4 fatty acid with a trans double bond. We presume this to have been formed from ct-18:2 (5). 4. In the liver lipids the fraction of 8,11,14-20:3, the arachidonate precursor was not affected by dietary ct,tc-18:2 (fig.4B). 5. Increasing dietary levels of ct,tc-18:2 caused decreases in the fractions of oleate and of 5,8,11-20:3 in the liver lipids, pre sumably because the ct,tc-18:2 was added to the diets at the expense of oleate. This is the same effect mentioned above as having been produced by tt-18:2. The ct-18:2 and tc-18:2 isomers gave different patterns of deposition in the tis sues ( table 4 ). In epididymal fat pads, the two isomers appeared in the same ratio as in the diet. But in liver the ratio of ct to tc was much lower than that in the diet, indi cating that although the tc isomer was de posited freely in the liver, the et isomer was almost completely excluded.
DISCUSSION When the mono-Ã-raiw and di-trans isomers of 18:2 are fed to rats, they differ from each other in their influences on the deposition of fatty acids in tissue lipids and on the fatty acid chain-elongation reactions. Ct,tc-l8:2 ¡nDiel Tissue deposition. The epididymal fat Fig. 4 The effects of dietary ct,tc-18:2 on the pads showed no selectivity for the deposi concentrations of the 18:2 isomers (A) and Cantion of trans fatty acids. The fat pads con polyenoic acids (B) in rat liver fatty acids. Each tained all of the trans isomers of 18:2 in point represents the mean ±standard error of the proportion to their concentrations in the mean for five animals.
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ratio of tt-18:2 to cc-18:2 in the liver lipids did not approach the composition of the dietary fat, for at the highest dietary level of tt-18:2, the tt/cc ratio in the diet was 7, but the tt/cc ratio in the liver was only 0.5. 3. The fraction of arachidonate in the liver lipids decreased with increasing levels of tt-18:2 in the diet. The change was only a slight one when the tt-18:2 level went from 9 to 19% (fig. 3B). 4. The fraction of 8,11,14-20:3 in the liver lipids was not affected by feeding tt-18:2 (fig. 3B). This observation is of interest because 8,11,14-20:3 is an inter mediate in the conversion of cc-18:2 to arachidonate. 5. The fractions both of oleate and of 5,8,11-20:3, its chain elongation product, in the liver lipids decreased with increasing levels of tt-18:2 in the diet. These decreases can be ascribed to the fact that the tt-18:2 was added to the diets at the expense of oleate. The following changes were produced by feedingct,tc-18:2. 1. In the epididymal fat pads the frac tions of both ct-18:2 and tc-18:2 increased with increasing levels of ct,tc-18:2 in the diet, while the fraction of cc-18:2 stayed constant (fig. 2B). This corresponds to the effect seen with tt-18:2. 2. In the liver lipids, the fraction of ct-18:2 increased slightly, and the fraction of tc-18:2 increased markedly as the level of ct,tc-18:2 increased in the diet, but the fraction of cc-18:2 was unchanged (fig. 4A).
397
OF irans-LINOLEATE
398
ANDERSON,
FULLMER
C\J 00
I
Control
ct,tc-ia-2 U-I8:2
0
2 Liver
4
6
cc-l8:2/Liver
8
10 °°
trons-l8;2
Fig. 5 Effects of the presence of the trans isomers of 18:2 on the yield of 20:4 from cc-18:2. (•) data from animals fed tt-18:2 diets and (A) data from animals fed ct,tc-18:2 diets. Each point is the mean ±standard error of the mean for five animals.
TABLE 5 Ratios of arachidonate to cc-18'.2 and of trans-20\4 to ct-18'.2 in the livers of control rats and of rats fed cl,tc-18:2 Fatty acid ratios in liver lipid1 Diet
¡rans-20:4/ct-18:2
Arachidonate/ co-18:2
Controlct,tc-18:2diet
±0.132.09 Cdiet ±1.062.52 ±0.092.25 ±0.092.46 ±0.412.34 Ddiet ±0.152.53 ±0.13 E2.90
1 Mean ±SEMfor five animals per treatment.
of 18:2 in almost the same proportion as they were fed. Thus, the specificity for 18:2 isomer accumulation is more a matter of the type of lipid being synthesized than of the tissue in which the synthesis occurs. Chain elongation. Dietary tt-18:2 re duced the concentration of 20:4 in the liver lipids (fig. 3), but increased the concentra tion of cc-18:2, which is its precursor (17). This apparent inhibition of 20:4 synthesis was proportional to the dietary level of tt-18:2, up to the point (9% tt-18:2) where the concentrations of cc-18:2 and 20:4 in the liver lipids were equal. A fur ther doubling of the tt-18:2 in the diet had no further effects on the concentrations of cc-18:2 or 20:4, even though the level of tt-18:2 in the liver lipids increased sub stantially. The influence of tt-18:2 on arachidonate synthesis is shown in figure 5. When the ratio of cc-18:2 to tt-18:2 in the liver was low (i.e., a low ratio of substrate to inhibitor ), then the ratio of arachidonate to cc-18:2 (i.e., product to substrate ratio) was low compared with the arachidonate/ cc-18:2 ratio seen at higher substrate/in hibitor ratios. The inhibition of arachidonate synthesis by tt-18:2 apparently happens early in the reaction sequence that converts cc-18:2 to 20:4 (17). The liver lipids of animals fed tt-18:2 showed no appreciable increase in 8,11,14-20:3, which is an intermediate in the synthesis of 20:4 from cc-18:2. The other possible intermediates (6,9,12-18:3 or 11,14-20:2) in the chain elongation sequence could not be detected at all. Thus, the supposed intermediates did not accumulate, but there was a copious in crease in starting material (cc-18:2). 6Anderson, R. L. Unpublished data.
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diet, and all of the isomers reached the same tissue concentration at any given dietary concentration. This observation agrees with the report of Beare-Rogers (16) that rats fed hydrogenated corn oil deposited lipoxidase-inactive 18:2 (prob ably trans isomers) in adipose tissue, al though not in liver. In contrast, the isomers of 18:2 differ in the degree of their incorporation into liver lipids, with cc-18:2 being more readily de posited than tc-18:2 which, in turn, is more readily deposited than tt-18:2. Even the tt-18:2, however, is deposited at levels proportional to its concentration in the diet. The ct-18:2 is almost completely excluded from deposition in the liver. The differences in deposition patterns for the 18:2 isomers between liver and adipose tissue reflect differences in the types of lipids in these tissues. The liver lipids con tained about 65% phospholipid, and it was found in previous work 5 that ct-18:2 was incorporated into liver phospholipids to only a slight extent, even though the neutral lipids of liver contained the isomers
AND HOLLENBACH
METABOLIC
EFFECTS
399
_ 0.2 00
o> > rO
co io
®Control 4 ct,tc-l8s2 .
00
tt-l8:2
25 50 75 oo Liver l8-l/trons-18-2
Fig. 6 Effects of the liver concentrations of the irons isomers of 18:2 on the yield of 5,8,1120:3 from liver 18:1. Data are expressed as ratios of liver 5,8,ll-20:3/liver 18:1 as a function of liver 18:I/liver irans-18:2. (•) data from ani mals fed tt-18:2 and (A) data from animals fed ct,tc-18:2. Each point is the mean ± standard error of the mean for five animals.
United States in 1965, according to Call and Sanchez (19). At the same time, hydrogenated fats contributed polyunsatu rated fatty acids (measured oefore hy drogénation) in an amount equal to 10% of the total fatty acids. If all of the poly unsaturated fatty acids that were exposed to hydrogénation had been converted to tt-18:2, then the ratio of cc-18:2 to tt-18:2 in the disappearing oils would have been about 1.2. This ratio corresponds to the cc-18:2/tt-18:2 ratio of 1.15, which was fed as tt-18:2, diet B, of our experiment. This ratio had very little effect on the fatty acid composition of liver lipids (fig. 3) or on the conversion of cc-18:2 to 20:4 (fig. 5). But in reality, hydrogénationdoes not result in substantial conversion of cc-18:2 to iraas-18:2. The fraas-18:2 isomers in most hydrogenated fats amount to no more than 5% of the total fatty acids (20-22) and less than 10% of the total 18:2. And of the total frans-18:2 in hydrogenated fat,
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Therefore, tt-18:2 must compete with cc18:2 at the first step of 20:4 synthesis, probably the desaturation of cc-18:2 to 6,9,12-18:3 (18). This observation agrees with the results of Brenner and Peluffo (7) which showed that tt-18:2 inhibits 18:2 conversion to y-linolenic acid by liver microsomes. The mono-fraas isomer mixture behaved quite differently. It did not inhibit 20:4 synthesis (fig. 4), and its presence did not alter the arachidonate/cc-18:2 ratio in the liver (fig. 5). The ct, tc-18:2 mixture did itself give rise to a ira as 20:4 acid (fig. 4). The work of Privett et al. (5) indicated that, of the two mono-irons isomers, only ct-18:2 functions as a precursor for chain elongation. Table 5 shows the ratios of C2o to Gig polyunsaturated fatty acids in the liver lipids of the animals. The ratio of trans-2Q:4 to ct-18:2 was the same as the ratio of arachidonate to cc-18:2 irrespective of the level of ct-18:2 in the diet. From this we infer that ct-18:2 is elongated to trans-20:4 as readily as cc-18:2 is elongated to arachidonate. But the constancy of the ratio also indicates that ct-18:2 does not markedly interfere with or suppress the synthesis of arachidonate from cc-18:2. Although the various trails isomers had different effects on 20:4 synthesis, both the di-trans isomer and the mono-iraus mixture reduced the concentration of 5,8,11-20:3 in liver lipids (figs. 3 and 4). Figure 6 indi cates that this effect was not solely a func tion of the reduced levels of 18:1 in the diet. The plot shows that the triene/monoene ratio in the liver decreased as the monoene/trans 18:2 ratio decreased. Thus the trans-18:2 isomers inhibited the syn thesis of 5,8,11-20:3. Similar effects were exhibited by both the mono-fraas mixture and the à i-trans 18:2 isomer. Dietary implications. We have shown that arachidonate synthesis can be partially inhibited by feeding a diet that contains a high ratio of tt-18:2 to cc-18:2, even in rats that do not have a deficiency of essen tial fatty acid. It is important to recognize the extent to which the diet that produced this effect differs from practical human or animal diets. Polyunsaturated fatty acids from nonhydrogenated sources accounted for 12% of the total fatty acids disappearing in the
OF irorw-LINOLEATE
400
ANDERSON, FULLMER AND HOLLENBACH
10. 11. 12.
ACKNOWLEDGMENTS
Dale Stitzel and Charles Allen developed and executed the gas Chromatographie pro cedures for analyzing and separating the isomerie fatty acids. Neil Artman contrib uted to the preparation of the manuscript. LITERATURE CITED 1. Selinger, Z. & Holman, R. T. (1965) The effects of trans, irans-linoleate upon the me tabolism of linoleate and linolenate and the positional distribution of linoleate isomers in liver lecithin. Biochim. Biophys. Acta 106, 56-62. 2. Kiinura, Y. & Tsuchiya, F. (1966) Effect of linoelaidic acid upon linoleic and oleic acid metabolism. Ann. Paediat. Jap. 12, 101-110. 3. Tsuchiya, F. & Kimura, Y. (1967) Further report on the effects of linoelaidic acid upon the metabolism of linoleic and oleic acid. Agr. Biol. Chem. 31, 5A-6A. 4. Prive«,O. S. & Blank, M. L. ( 1964) Studies on the metabolism of linoelaidic acid in the essential fatty acid-deficient rat. J. Amer. Oil Chem. Soc. 41, 292-297. 5. Prive«, O. S., Stearns, E. M., Jr. & Nickell, E. C. (1967) Metabolism of the geometrie isomers of linoleic acid in the rat. J. Nutr. 92, 303-310. 6. Williams, M. A., Tamai, K. T., Hincenbergs, I. & Mclntosh, D. J. (1972) Hydrogenated coconut oil and tissue fatty acids in EFAdepleted and EFA-supplemented rats. J. Nutr. 102, 847-855. 7. Brenner, R. R. & Peluffo, R. O. (1969) Regulation of unsaturated fatty acid biosyn thesis. I. Effect of unsaturated fatty acid of 18 carbons on the microsomal desaturation of linoleic acid to 7-linolenic acid. Biochim. Bio phys. Acta 176, 471-479. 8. Knipprath, W. G. & Mead, J. F. (1964) The metabolism of Ã-rans,Ã-raris-octadecadienoic acid. Incorporation of rrans,trans-octadecadienoic acid into the Cx polyunsaturated acids of the rat. J. Amer. Oil Chem. Soc. 41, 437440. 9. Mattson, F. H. (1960) An investigation of the essential fatty acid activity of some of
13.
14.
15. 16.
17. 18.
19.
20. 21.
22.
23.
24.
the geometrical isomers of unsaturated fatty acids. J. Nutr. 71, 366-370. Olivecrona, T. (1962) The metabolism of l-C"-palmitic acid in the rat. Acta Physiol. Scand. 54, 295-305. Carroll, K. K. (1961) Separation of lipid classes by chromatography on Fiorisi!. J. Lipid Res. 2, 135-141. Kuemmel, D. F. & Chapman, L. R. (1966) Analysis of methyl octadecenoate and octadecadienoate isomers by combined liquidsolid and capillary gas—liquid. Anal. Chem. 38, 1611. Downing, D. T. & Greene, R. S. (1968) Rapid determination of double-bond positions in monoenoic fatty acids by periodate—permanganate oxidation. Lipids 3, 96-100. Coots, R. H. (1964) A comparison of the metabolism of cis,cts-linoleic, tran$,trans-linoleic, and a mixture of cis,trans- and trans,cislinoleic acids in the rat. J. Lipid Res. 5, 473476. Snedecor, G. W. (1956) Statistical Methods, pp. 237-290, The Iowa State College Press, Ames. Beare-Rogers, J. L. (1970) The deposition of polyunsaturated fatty acids in the rat fed partially hydrogenated vegetable oil. J. Amer. Oil Chem. Soc. 47, 487-489. Mead, J. F. (1961) Synthesis and metab olism of polyunsaturated acids. Federation Proc. 20, 952-955. Mohrhauer, H., Christiansen, K., Can, M. V., Dewbig, M. & Holman, R. T. ( 1967) Chain elongation of linoleic acid and its inhibition by other fatty acids in vitro. J. Biol. Chem. 242, 4507-1514. Call, D. L. & Sanchez, A. M. (1967) Trends in fat disappearance in the United States 1909-1965. J. Nutr. Suppl. I, Part II, vol. 93, no. 2. Carpenter, D. L. & Slover, H. T. (1973) Lipid composition of selected margarines. J. Amer. Oil Chem. Soc. SO, 372-376. Hoelmer, G. & Aaes-Joergensen, E. (1969) Essential fatty acid deficient rats. II. Fatty acid composition of partially hydrogenated arachis, soybean and herring oil and normal refined arachis oil. Lipids 4, 507-514. Scholfield, C. R., Davison, V. L. & Dutton, H. T. (1967) Analysis of geometrical and positional isomers of fatty acids in partially hydrogenated fats. J. Amer. Oil Chem. Soc. 44, 648-651. Williams, M. A. & Briggs, G. M. (1963) An evaluation of mineral mixtures commonly used in diets of experimental animals. Amer. J. Clin. Nutr. 13, 115-121. Carroll, K. K. (1963) Acid-treated Florisil as an absorbent for column chromatography. J. Amer. Ou Chem. Soc. 40. 413-419.
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only a small fraction is the tt-18:2, which inhibited arachidonate synthesis. Thus, it is apparent that the high levels of dietary tt-18:2 required to reduce cc-18:2 conver sion to 20:4 can be attained only by the use of deliberately isomerized fats and can not be attained from partially hydrogenated fats.
KEY WORDS
linoleic acid metabolism
The geometrical isomers of unsaturated fatty acids occur widely. Because they are formed by the partial hydrogénation of polyunsaturated fats in the rumens of sheep and cattle, they appear in the milk and body fats of these animals. Because they are formed by the partial hydrogénationof polyunsaturated fats, they appear in commereiai shortenings and margarines. Because of their evident importance, a substantial body of information has been built up about the digestion, absorption, and catabolism
of both
trans-l8:l
and
ienmpr« nf linnlpiri IK .«i 01 tne trans isomers or nnoieic
acid
(18:2) on the conversion , i, . ' . j , nr. .* , arachldoniC acid (20:4) have
OÕ 18:2 tO , , j. j been Studied
in both the intact animal (1-6) and at the enzyme level (7). In the intact animal the io n • 18:2 ISOmer
-it With
two
trans
i ui double
•arachi-
inhibits 20:4 synthesis (1-5), but the 18:2 isomers with one trans bond do not in fluence 20:4 synthesis ( 1 ). It has also been shown that the 18:2 isomer with two trans bond inhibits the microsomal enzyme system that converts linoleic acid to y-linolenic acid (7). It has also been demonstrated that ct-18:2 but not tc-18:2 can be converted to a 20:4 with a trans double bond (5). There exists some controversy as to •
tnns-
io n r u • J lo:Z tatty aCIUS. Tbp pflW'ts nf tVip franc
•trans fatty acids
Receivedfor publication June 20.1974. Abbreviations : in the designation of the geometric Isomers of linoleic acid, the first letter refers to the configuration of the double bond at the 9,10-posltion
nnd the gecondletter refers to the configurationof the double bond at the 12.13-posltlon ; c = ci»; t = trans. For example. ct-18:2 = 9-CÃ-»,12-Ã-rThe ct,tc-18:2 used to prepare these diets had a ct/tc ratio of 0.6.
whether tt-18:2 can be converted to a 20:4 acid with two trans bonds. Knipprath and Mead (8) have presented evidence that tt-18:2 is elongated to 20:4, while Privett et al. (5) and Kimura and Tsuchiya (2) observed no 20:4 with irons bonds when tt-18:2wasfed. In the work reported here, the 18:2 isomers with one or two trans bonds were fed to rats at graded levels with a constant supply of cc-18:2 to ascertain the level of trans-18:2 required to influence the con version of cc-18:2 to 20:4. The report also describes the liver and adipose tissue con centrations of the 18:2 isomers as a func tion of dietary level. TABLE 2 Composition of the diets
Casein Sucrose Water-soluble vitamins in sucrose1 Salt mixture U.S.P. XIV2 Cellulose3
Fat-soluble vitamin mixture4
Fat
18
M 5 4
:< l
19
100 1 The water-soluble vitamin mixture containined (in mg) : menadiune, 0.3: thiamin-HCl, 0.4; riboflavin, 0.7: niacin, 2.0; calcium pantothenate, 0.2; folie acid, 0.002; pyridoxine, 0.4; vitamin B-12, 0.015; choline chloride, 300; inositol, 200; ascorbic acid, 10 ; biotin, 0,015 ; p-aminobenzoic acid, 10. 1 Nutritional Biochemicals, Inc., Cleveland, Ohio (23). * Celluflour, Chicago Dietetic Supply House, Chicago, 111. ' The fat-soluble vitamin mixture was prepared using the appropriate fat for the diet. It contained (in U.S.P. units) : retinyl acetate, 1,250; ergocaleiferol, (1,250); o-a-tocopherol, 10.
MATERIALS AND METHODS
Preparation of fats. Pure methyl linoleate was obtained by low temperature urea ad duction of safflower oil methyl esters. Treatment with SOL>converted it to a mix ture of geometrical isomers (9). Low tem perature crystallization yielded two frac tions; one was the isomer with two trans bonds (tt-18:2), and the other was a mix ture of the isomers with one trans bond ( ct,tc-18:2 ). Analysis by argentation thinlayer chromatography (4) showed the presence of a small amount of mono-irons material as a contaminant in the tt-18:2 preparation, while the ct,tc-18:2 prepara tion contained only traces of the di-trans isomer. The methyl esters were transesterified with glycerol and alkaline catalyst to con vert them to triglycérides,each containing a single fatty acid. These were mixed with other pure triglycéridesin chosen propor tions, and the mixtures were randomized by treatment with sodium methoxide under nitrogen. The resulting triglycéridesconsti tuted a series having trans-18:2/cc-18:2 rations of 0.5, 1, 2, 4, and 8, with each product containing 2.5% of cc-18:2. Thus, as shown in table 1, several levels of tt-18:2 or of ct,tc-18:2 were added to the diet at the expense of 18:1. The triglyc éridesthat contained ct,tc-18:2 were made from two different preparations of this isomer mixture, and they differed in their ct/tc ratios as shown in table 1.
3(J5
METABOLIC EFFECTS OF iraus-LINOLEATE 5,8,11,14-20:4
5,8,11-20:3 8,11,14-20:3
B.
trans-20:4?
80
82 Time(min.)
84
86
Fig. 1 Tracings of Cao-polyenoic acid region of capillary chromatographs of liver lipid fatty acid methyl esters from animals fed (A) control diet; (B) tt-18:2, diet E (table 1); and (C) ct,tc-18:2, diet E (table 1).
lary GLC column. Retention times of their components were then compared with re tention times that had been established for some specific structures. Knowledge of double bond positions came (13) by application of Downing and Greene's cleavage technique. The trans bond content of the isolated 20:4 was determined by infrared spectroscopy (14). One analytical ambiguity remained. The capillary GLC column did not separate tc from tt-18:2, and therefore the composi tion of this peak in the samples of animal lipid could not be established by GLC evidence alone. But it was known from argentation TLC that the di-trans product contained only a small amount of monotrans material, and that the mono-trans mixture contained very little di-trans con taminant. Because the materials being fed were thus clearly defined, and because only peaks with the same retention time as those in the dietary fat fed were observed, we re3Partition liquid and solid support were obtained from Applied Science Laboratories, State College, Pa. « Hewlett-Packard model 720.
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Feeding experiment. The triglycérides were incorporated into semisynthetic diets at a level of 20% (table 2). The diets were fed to weanling, male, Sprague-Dawley rats (five rats per diet) for 6 weeks. The rats were housed individually in cages with raised wire floors. The animals were housed in a room with constant temperature (25°) and humidity (52% of saturation) and a 12 hour light-dark cycle. Fresh feed was provided twice per week, and body weights were determined each week. The animals were killed after consuming the diets for 6 weeks, and their livers and epididymal fat pads were excised, rapidly weighed, frozen, and stored at —20° until analyzed. Total lipid extracts of the individual livers and of the pooled fat pads from each di etary group were prepared (10). Methyl esters of the fatty acids were prepared by refluxing the total lipid extracts with 2% H^SO4 in methanol in a nitrogen atmo sphere. The methyl esters were freed of cholesterol by chromatography on Florisil (11). Fatty acid analyses. The fatty acid com positions of the lipid materials were deter mined by gas-liquid chromatography (GLC) of their methyl esters. Separations were made on a 10 foot X 1/4 inch stain less steel column packed with 15% EGSS-X on 60/80 mesh Gas-Chrom P.3 Thermalconductivity gas chromatography was used with the following operating conditions: column oven temperature 190°,injection port 330°, detector 350°,flow rate 79.5 ml/minute.4 The 18:2 isomers and the C2o-polyenoic acid isomers were analyzed on a polyphenyl ether capillary column as described by Kuemmel and Chapman (12). The column was held at 180°,and the inlet pressure was increased from 30 to 60 psi at 1 hour ( after elution of the 18:2 isomers) to sharpen resolution of the C20 peaks. On this column it was possible to separate the 18:2 esters into cc-18:2, ct-18:2, and a mixture of tc-18:2 and tt-18:2. Figure 1 shows some separations of the C20-fatty acid esters on the capillary col umn. As indicated on the graph, some of the individual components were identified. This was done by collecting the 20:3 and 20:4 esters from the packed GLC column and rechromatographing them on the capil-
390
ANDERSON,
FULLMER
AND HOLLENBACH
TABLE 3 Amount and phoapholipid content of lipid in the livers of rats fed trans isomers of linoleic acid Diet
Weight
Epididymal fat pad1
Controltt-18:2 diet Adiet Bdiet Cdiet Ddiet Ect,tc-18:2 diet Adiet Bdiet Cdiet Ddiet Ea252
Liver lipids
a'4.2±0.2°4.0±0.1°4.1±0.1«3.8 100 phospholipid*63
±1°66 ±10«278±10°251 ±0.1«2.1 ±1°66±2°64±1«64±1«63±1 8"251 ± ±0.1«1.8±0.2°2.0 7°243 ± ±0.3°4.5 4°262 ± ±0.1«2.1 ±0.3«5.3 ±0.2«1.8±0.2°2.3±0.1°2.2±0.1°2.1 ±0.3»4.6±0.2«4.5 7°247 ± 5«251 ± ±10°248 ±0.2°4.5±0.5°4. ±1°65 ±12"257 ±1°64±1°63 5 ±10°255 ±0.1«2.2±0.1«gl±0.4"4.6±0.2°% ±1« ± 4«al
1 Tissue weight g/100 g body weight. Mean ±-IM of five animals. ' Grains of lipid per 100 g of liver. Mean ±SEMfor five animala per treatment ; numbers with same letter superscript are not significantly different from each other. * Percent phospho lipid determined by chromatography on acid-washed Fiorisi! (24).
ported the equivocal peak as tc-18:2 when it occurred in the lipids of animals fed ct,tc-18:2, and as tt-18:2 when it occurred in the lipids of animals fed tt-18:2. Statistical analyses employed analyses of variance as outlined by Snedecor (15).
affected the fraction of phospholipid in the liver lipids. Feeding the trans isomers to rats influ enced the compositions of their liver and depot lipids. The following changes were produced by the tt-18:2.
RESULTS None of the 11 diets produced any sig nificant differences in body or epididymal fat pads weights. As shown in table 3, the liver lipid content was slightly, but not significantly, elevated in all of the groups fed ct,tc-18:2. Among animals receiving tt-18:2, the liver lipid content was not sig nificantly elevated above the control level except in the animals fed the highest level of tt-18:2. None of the dietary treatments
1. In the epididymal fat pads, the frac tion of tt-18:2 increased with increasing levels of tt-18:2 in the diet, while the frac tion of cc-18:2 stayed constant (fig. 2A). 2. In the liver lipids, the fraction of tt18:2 increased with increasing levels of tt-18:2 in the diet. The fraction of cc-18:2 also increased up to the point where the diet contained 9% tt-18:2 and then re mained constant (fig. 3A). Even so, the
All t_u 20 4
».II.U-20,»
0
5
10 15 20 % 11-18:2in tht Ditl
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g2.0±0.2°2.1 100
5 O 15 20 let,te -18:2 in the Di«
Fig. 2 The effects of dietary tt-18:2 (A) and ct,tc-18:2 (B) on the 18:2 geometric isomer con centrations in epididymal fat pad fatty acids. Obtained on epididymal fat pads pooled from five animals per treatment.
% 11-18:2 i" Ihr D«!
Fig. 3 The effects of dietary tt-18:2 on the concentrations of the 18:2 isomers (A), and G»polyenoic acids (B) in rat liver fatty acids. Each point represents the mean ±standard error of the mean for five animals.
METABOLIC
EFFECTS
TABLE 4 Ratios of ct-18:e lo tc-18:2 in the diets, livers, and adipose tissues of rats fed diets containing various levels of these isomers Diet
Liver
Adipose tissue
ct-lS-.Z/tc-lS'.Z10.34
1.21'0.613Ratio ±0.07(10) 0.17±0.01(15)1.282
0.613
1 Mean ±SEM;numl>er of samples indicated in parentheses. ! Animals fed oils ct,tc-18:2, diets A and U (see table 1). • Animals fed oils ct,tc-18:2, diets C, D, and E (table 1).
3. The fraction of arachidonate in the liver lipids was not affected by additions of ct,tc-18:2 to the diet (fig. 4B). The dietary ct,tc-18:2 did give rise to increasing levels of a fatty acid that was identified (by capillary GLC retention time and IR) as a 20:4 fatty acid with a trans double bond. We presume this to have been formed from ct-18:2 (5). 4. In the liver lipids the fraction of 8,11,14-20:3, the arachidonate precursor was not affected by dietary ct,tc-18:2 (fig.4B). 5. Increasing dietary levels of ct,tc-18:2 caused decreases in the fractions of oleate and of 5,8,11-20:3 in the liver lipids, pre sumably because the ct,tc-18:2 was added to the diets at the expense of oleate. This is the same effect mentioned above as having been produced by tt-18:2. The ct-18:2 and tc-18:2 isomers gave different patterns of deposition in the tis sues ( table 4 ). In epididymal fat pads, the two isomers appeared in the same ratio as in the diet. But in liver the ratio of ct to tc was much lower than that in the diet, indi cating that although the tc isomer was de posited freely in the liver, the et isomer was almost completely excluded.
DISCUSSION When the mono-Ã-raiw and di-trans isomers of 18:2 are fed to rats, they differ from each other in their influences on the deposition of fatty acids in tissue lipids and on the fatty acid chain-elongation reactions. Ct,tc-l8:2 ¡nDiel Tissue deposition. The epididymal fat Fig. 4 The effects of dietary ct,tc-18:2 on the pads showed no selectivity for the deposi concentrations of the 18:2 isomers (A) and Cantion of trans fatty acids. The fat pads con polyenoic acids (B) in rat liver fatty acids. Each tained all of the trans isomers of 18:2 in point represents the mean ±standard error of the proportion to their concentrations in the mean for five animals.
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ratio of tt-18:2 to cc-18:2 in the liver lipids did not approach the composition of the dietary fat, for at the highest dietary level of tt-18:2, the tt/cc ratio in the diet was 7, but the tt/cc ratio in the liver was only 0.5. 3. The fraction of arachidonate in the liver lipids decreased with increasing levels of tt-18:2 in the diet. The change was only a slight one when the tt-18:2 level went from 9 to 19% (fig. 3B). 4. The fraction of 8,11,14-20:3 in the liver lipids was not affected by feeding tt-18:2 (fig. 3B). This observation is of interest because 8,11,14-20:3 is an inter mediate in the conversion of cc-18:2 to arachidonate. 5. The fractions both of oleate and of 5,8,11-20:3, its chain elongation product, in the liver lipids decreased with increasing levels of tt-18:2 in the diet. These decreases can be ascribed to the fact that the tt-18:2 was added to the diets at the expense of oleate. The following changes were produced by feedingct,tc-18:2. 1. In the epididymal fat pads the frac tions of both ct-18:2 and tc-18:2 increased with increasing levels of ct,tc-18:2 in the diet, while the fraction of cc-18:2 stayed constant (fig. 2B). This corresponds to the effect seen with tt-18:2. 2. In the liver lipids, the fraction of ct-18:2 increased slightly, and the fraction of tc-18:2 increased markedly as the level of ct,tc-18:2 increased in the diet, but the fraction of cc-18:2 was unchanged (fig. 4A).
397
OF irans-LINOLEATE
398
ANDERSON,
FULLMER
C\J 00
I
Control
ct,tc-ia-2 U-I8:2
0
2 Liver
4
6
cc-l8:2/Liver
8
10 °°
trons-l8;2
Fig. 5 Effects of the presence of the trans isomers of 18:2 on the yield of 20:4 from cc-18:2. (•) data from animals fed tt-18:2 diets and (A) data from animals fed ct,tc-18:2 diets. Each point is the mean ±standard error of the mean for five animals.
TABLE 5 Ratios of arachidonate to cc-18'.2 and of trans-20\4 to ct-18'.2 in the livers of control rats and of rats fed cl,tc-18:2 Fatty acid ratios in liver lipid1 Diet
¡rans-20:4/ct-18:2
Arachidonate/ co-18:2
Controlct,tc-18:2diet
±0.132.09 Cdiet ±1.062.52 ±0.092.25 ±0.092.46 ±0.412.34 Ddiet ±0.152.53 ±0.13 E2.90
1 Mean ±SEMfor five animals per treatment.
of 18:2 in almost the same proportion as they were fed. Thus, the specificity for 18:2 isomer accumulation is more a matter of the type of lipid being synthesized than of the tissue in which the synthesis occurs. Chain elongation. Dietary tt-18:2 re duced the concentration of 20:4 in the liver lipids (fig. 3), but increased the concentra tion of cc-18:2, which is its precursor (17). This apparent inhibition of 20:4 synthesis was proportional to the dietary level of tt-18:2, up to the point (9% tt-18:2) where the concentrations of cc-18:2 and 20:4 in the liver lipids were equal. A fur ther doubling of the tt-18:2 in the diet had no further effects on the concentrations of cc-18:2 or 20:4, even though the level of tt-18:2 in the liver lipids increased sub stantially. The influence of tt-18:2 on arachidonate synthesis is shown in figure 5. When the ratio of cc-18:2 to tt-18:2 in the liver was low (i.e., a low ratio of substrate to inhibitor ), then the ratio of arachidonate to cc-18:2 (i.e., product to substrate ratio) was low compared with the arachidonate/ cc-18:2 ratio seen at higher substrate/in hibitor ratios. The inhibition of arachidonate synthesis by tt-18:2 apparently happens early in the reaction sequence that converts cc-18:2 to 20:4 (17). The liver lipids of animals fed tt-18:2 showed no appreciable increase in 8,11,14-20:3, which is an intermediate in the synthesis of 20:4 from cc-18:2. The other possible intermediates (6,9,12-18:3 or 11,14-20:2) in the chain elongation sequence could not be detected at all. Thus, the supposed intermediates did not accumulate, but there was a copious in crease in starting material (cc-18:2). 6Anderson, R. L. Unpublished data.
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diet, and all of the isomers reached the same tissue concentration at any given dietary concentration. This observation agrees with the report of Beare-Rogers (16) that rats fed hydrogenated corn oil deposited lipoxidase-inactive 18:2 (prob ably trans isomers) in adipose tissue, al though not in liver. In contrast, the isomers of 18:2 differ in the degree of their incorporation into liver lipids, with cc-18:2 being more readily de posited than tc-18:2 which, in turn, is more readily deposited than tt-18:2. Even the tt-18:2, however, is deposited at levels proportional to its concentration in the diet. The ct-18:2 is almost completely excluded from deposition in the liver. The differences in deposition patterns for the 18:2 isomers between liver and adipose tissue reflect differences in the types of lipids in these tissues. The liver lipids con tained about 65% phospholipid, and it was found in previous work 5 that ct-18:2 was incorporated into liver phospholipids to only a slight extent, even though the neutral lipids of liver contained the isomers
AND HOLLENBACH
METABOLIC
EFFECTS
399
_ 0.2 00
o> > rO
co io
®Control 4 ct,tc-l8s2 .
00
tt-l8:2
25 50 75 oo Liver l8-l/trons-18-2
Fig. 6 Effects of the liver concentrations of the irons isomers of 18:2 on the yield of 5,8,1120:3 from liver 18:1. Data are expressed as ratios of liver 5,8,ll-20:3/liver 18:1 as a function of liver 18:I/liver irans-18:2. (•) data from ani mals fed tt-18:2 and (A) data from animals fed ct,tc-18:2. Each point is the mean ± standard error of the mean for five animals.
United States in 1965, according to Call and Sanchez (19). At the same time, hydrogenated fats contributed polyunsatu rated fatty acids (measured oefore hy drogénation) in an amount equal to 10% of the total fatty acids. If all of the poly unsaturated fatty acids that were exposed to hydrogénation had been converted to tt-18:2, then the ratio of cc-18:2 to tt-18:2 in the disappearing oils would have been about 1.2. This ratio corresponds to the cc-18:2/tt-18:2 ratio of 1.15, which was fed as tt-18:2, diet B, of our experiment. This ratio had very little effect on the fatty acid composition of liver lipids (fig. 3) or on the conversion of cc-18:2 to 20:4 (fig. 5). But in reality, hydrogénationdoes not result in substantial conversion of cc-18:2 to iraas-18:2. The fraas-18:2 isomers in most hydrogenated fats amount to no more than 5% of the total fatty acids (20-22) and less than 10% of the total 18:2. And of the total frans-18:2 in hydrogenated fat,
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Therefore, tt-18:2 must compete with cc18:2 at the first step of 20:4 synthesis, probably the desaturation of cc-18:2 to 6,9,12-18:3 (18). This observation agrees with the results of Brenner and Peluffo (7) which showed that tt-18:2 inhibits 18:2 conversion to y-linolenic acid by liver microsomes. The mono-fraas isomer mixture behaved quite differently. It did not inhibit 20:4 synthesis (fig. 4), and its presence did not alter the arachidonate/cc-18:2 ratio in the liver (fig. 5). The ct, tc-18:2 mixture did itself give rise to a ira as 20:4 acid (fig. 4). The work of Privett et al. (5) indicated that, of the two mono-irons isomers, only ct-18:2 functions as a precursor for chain elongation. Table 5 shows the ratios of C2o to Gig polyunsaturated fatty acids in the liver lipids of the animals. The ratio of trans-2Q:4 to ct-18:2 was the same as the ratio of arachidonate to cc-18:2 irrespective of the level of ct-18:2 in the diet. From this we infer that ct-18:2 is elongated to trans-20:4 as readily as cc-18:2 is elongated to arachidonate. But the constancy of the ratio also indicates that ct-18:2 does not markedly interfere with or suppress the synthesis of arachidonate from cc-18:2. Although the various trails isomers had different effects on 20:4 synthesis, both the di-trans isomer and the mono-iraus mixture reduced the concentration of 5,8,11-20:3 in liver lipids (figs. 3 and 4). Figure 6 indi cates that this effect was not solely a func tion of the reduced levels of 18:1 in the diet. The plot shows that the triene/monoene ratio in the liver decreased as the monoene/trans 18:2 ratio decreased. Thus the trans-18:2 isomers inhibited the syn thesis of 5,8,11-20:3. Similar effects were exhibited by both the mono-fraas mixture and the à i-trans 18:2 isomer. Dietary implications. We have shown that arachidonate synthesis can be partially inhibited by feeding a diet that contains a high ratio of tt-18:2 to cc-18:2, even in rats that do not have a deficiency of essen tial fatty acid. It is important to recognize the extent to which the diet that produced this effect differs from practical human or animal diets. Polyunsaturated fatty acids from nonhydrogenated sources accounted for 12% of the total fatty acids disappearing in the
OF irorw-LINOLEATE
400
ANDERSON, FULLMER AND HOLLENBACH
10. 11. 12.
ACKNOWLEDGMENTS
Dale Stitzel and Charles Allen developed and executed the gas Chromatographie pro cedures for analyzing and separating the isomerie fatty acids. Neil Artman contrib uted to the preparation of the manuscript. LITERATURE CITED 1. Selinger, Z. & Holman, R. T. (1965) The effects of trans, irans-linoleate upon the me tabolism of linoleate and linolenate and the positional distribution of linoleate isomers in liver lecithin. Biochim. Biophys. Acta 106, 56-62. 2. Kiinura, Y. & Tsuchiya, F. (1966) Effect of linoelaidic acid upon linoleic and oleic acid metabolism. Ann. Paediat. Jap. 12, 101-110. 3. Tsuchiya, F. & Kimura, Y. (1967) Further report on the effects of linoelaidic acid upon the metabolism of linoleic and oleic acid. Agr. Biol. Chem. 31, 5A-6A. 4. Prive«,O. S. & Blank, M. L. ( 1964) Studies on the metabolism of linoelaidic acid in the essential fatty acid-deficient rat. J. Amer. Oil Chem. Soc. 41, 292-297. 5. Prive«, O. S., Stearns, E. M., Jr. & Nickell, E. C. (1967) Metabolism of the geometrie isomers of linoleic acid in the rat. J. Nutr. 92, 303-310. 6. Williams, M. A., Tamai, K. T., Hincenbergs, I. & Mclntosh, D. J. (1972) Hydrogenated coconut oil and tissue fatty acids in EFAdepleted and EFA-supplemented rats. J. Nutr. 102, 847-855. 7. Brenner, R. R. & Peluffo, R. O. (1969) Regulation of unsaturated fatty acid biosyn thesis. I. Effect of unsaturated fatty acid of 18 carbons on the microsomal desaturation of linoleic acid to 7-linolenic acid. Biochim. Bio phys. Acta 176, 471-479. 8. Knipprath, W. G. & Mead, J. F. (1964) The metabolism of Ã-rans,Ã-raris-octadecadienoic acid. Incorporation of rrans,trans-octadecadienoic acid into the Cx polyunsaturated acids of the rat. J. Amer. Oil Chem. Soc. 41, 437440. 9. Mattson, F. H. (1960) An investigation of the essential fatty acid activity of some of
13.
14.
15. 16.
17. 18.
19.
20. 21.
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only a small fraction is the tt-18:2, which inhibited arachidonate synthesis. Thus, it is apparent that the high levels of dietary tt-18:2 required to reduce cc-18:2 conver sion to 20:4 can be attained only by the use of deliberately isomerized fats and can not be attained from partially hydrogenated fats.
