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Effect of geometric isomers of δ9-octadecenoic acid on liver lipid biosynthesis by essential fatty acid deficient rats
411
Biochimica 0 Elsevier
et Biophysics
Scientific
Acta,
Publishing
369
(1974)
Company,
41 l-420
Amsterdam
- Printed
in The Netherlands
BBA 56513
EFFECT OF GEOMETRIC ISOMERS OF A’-OCTADECENOIC ACID ON LIVER LIPID BIOSYNTHESIS BY ESSENTIAL FATTY ACID DEFICIENT RATS L.S.S. GUO* and J.C. ALEXANDER Department
(Received
of Nutrition,
University
of Guelph,
Guelph,
Ontario
NlG
2Wl
(Canada)
May 9th, 1974)
Summary 1. Liver lipid biosynthesis was studied by measuring the incorporation of radioactivity 0.5 h after the administration of [l-i 4 C] acetate to rats. The animals were fed 10% fat diets containing corn oil, hydrogenated coconut oil, ethyl oleate, or ethyl elaidate. 2. Incorporation of labelled acetate into liver lipids was enhanced in the three linoleic acid deficient dietary groups (hydrogenated coconut oil, ethyl oleate and ethyl elaidate). The highest level of labelling was found when ethyl elaidate was the dietary fat. 3. Patterns of distribution of ’ 4 C among liver lipid classes were altered in the hydrogenated coconut oil and ethyl oleate dietary groups compared to the corn oil control group; the label was increased in the triacylglycerols, and decreased in the phospholipids. In the case of the ethyl elaidate group, in addition to the triacylglycerols, high labelling was shown in the phospholipids, the monoacylglycerols and the cholesterol esters. 4. In the animals fed the trans fatty acid, there was a significant increase in the radioactivity in liver palmitic, palmitoleic, steak, and oleic acids. Small concentrations of these radioactivities associated with the carboxyl carbons of the acids indicate that they are formed preferentially by de novo biosynthesis. 5. A relatively small increase in the specific activity of 20 : 3 compared to 18 : 1 in the truns acid-fed group reflects a slow conversion of 18 : 1 to 20 : 3. When the radioactivity in the carboxyl carbons of the 20 : 3 acid from the rats in each of the essential fatty acid deficient groups was compared, the lowest level was found with animals fed ethyl elaidate.
* Present 94120.
address: U.S.A.
Department
of
Nutritional
Science,
University
of
California.
Berkeley,
Calif.
412
Introduction It is well established that when animals are maintained on a diet deficient in essential fatty acid, levels of 5,8,11-eicosatrienoic acid and 4,7,10,13-eicosatetraenoic acid derived from ofeic acid and palmitoleic acid are elevated in tissue lipids [ 1,2]. These biosynthetic pathways, however, have been reported to be inhibited as a result of feeding trans fatty acids [3-61. When rats were fed a partially hydrogenated soybean fat containing trans fatty acids Egwim and Sgoutas [ 71 found that the metabolites from oleate and palmitoleate were accumulated as eicosadienoic acids in the liver lipids, with corresponding low levels of eicosatrienoic and eicosatetraenoic acids due to essential fatty acid deficiency. They suggested that the occurrence of these dienoic acids was due to changes in the activity of long-chain acyl desaturases in the liver microsomes. Evidence indicates that trans-octadecenoic acid can be desaturated in vivo to a 5c~s,9trans-o~tadecadienoic acid f6,8]. On the other hand, this trans monoene also is converted to stearic acid by a direct hydrogenation process [9]. In unpublished data from our laboratory, it was found that when rats were fed a 10% elaidate diet, in addition to the deposition of elaidic and 5cis,9transoctadecadienoic acids, a substantial amount of cis monounsaturated fatty acids was observed in liver phospholipids. It appeared that this natural cis tissue acid was being formed by (1) the endogenous biosynthesis from acetate, (2) the hydrogenation of dietary elaidic acid followed by desaturation, or (3) a combination of 1 and 2. The present experiment was designed to study the first of the above synthetic routes with incorporation of [l-’ 4 C] acetate into liver lipids by rats fed either cis or tram isomers of octadecenoic acid. Since tram fatty acids have been shown in many of their biological properties to be similar to saturated fatty acids, hydrogenated coconut oil was included in addition to corn oil as a control fat. Materials and Methods
Male weanling rats of the Wistar strain, obtained Guelph, Ontario, weighing 50-56 g were used.
from Woodlyn
Farms,
Preparation of ethyl esters For this study, the ethyl oleate was isolated and esterified from total fatty acids of olive oil by Iow temperature c~stallization process, followed by a urea treatment. The final material was 98% oleate ester with a small component of palmitoleic acid. Ethyl elaidate was prepared by isomerizing oleic acid with nitrous acid [lo] . One recrystallization from light petroleum produced 90% of the tram isomer. ~x~er~~entai procedure Male weanling rats were randomly distributed into four groups of 5 animals each, and maintained on diets containing: fat (lo%), dextrose (58X%), vitamin-free casein (ZO%), DL-methionine (0.2%), cellulose (5%), salt mix,
413
U.S.P. XIV (5%) and a vitamin mix (1%). The vitamin mixture contained the following per kilogram of diet: (in I.U.) vitamin A, 24 000; vitamin D, 4000; (in mg) vitamin E (as cw-tocopherol), 120: menadione, 1.0; thiamine - HCl, 10; pyridoxine * HCl, 5; niacin, 50; calcium pantothenate, 20; choline chloride, 1000; p-aminobenzoic acid, 20; inositol, 1000; riboflavin, 10; folic acid, 1.0; biotin, 0.5; (in pg) vitamin B1 *, 10. Corn oil, hydrogenated coconut oil, ethyl oleate and ethyl elaidate each served as a fat source in different dietary groups. The rats were fed the experimental diets for 12 weeks. During the last two weeks, the animals were mealfed only 2 h daily. Soon after the end of the feeding period on the last day of Corp., the experiment 25 /.K!i of sodium [l-l 4 C] acetate ( Amersham-Searle Toronto, Ontario) per 100 g of body weight was given to each rat by intraperitoneal injection. A preliminary experiment showed that the rate of incorporation of [’ 4 C] acetate into liver lipids of both corn oil and hydrogenated coconut oil-prefed rats was parallel within a 4 h interval following the injection of labelled compound. The peak of the incorporation was at 0.5 h after the administration. Therefore, this time period was selected for tissue collection. The animals were exsanguinated under diethyl ether anesthesia, their livers were removed, weighed and immediately homogenized. Lipid extraction
and fractionation
Lipids were extracted from the homogenized tissues three times with chloroform-methanol (2 : 1, v/v) by the procedure of Folch et al. [ 111. All combined extracts were analyzed for phospholipid content by the method of Bartlett [12]. A portion of total lipids was separated into different classes by thin-layer chromatography on Silica gel G. The plates were developed with light petroleum-diethyl ether-acetic acid (90 : 10 : 1, by vol.). Components were scraped from the plates and extracted three times into scintillation vials. For the extraction of the components of neutral lipids, diethyl ether was used, while the phospholipid fraction was extracted with methanol. The remaining portion of the lipids which was not used for thin-layer chromatography was interesterified with 14% borontrifluoride-methanol (w/v) under N 2 [ 131 and the methyl esters were separated from the free cholesterol by a silicic acid column. These fatty acid esters were fractionated by argentation thin-layer chromatography according to their degrees of unsaturation. The methyl ester fractions were further separated or purified by preparative gasliquid chromatography. An Aerograph Model 1520 Gas Chromatograph equipped with a thermal conductivity detector, and coiled aluminum columns (360 cm X 0.6 cm) was used. The columns were packed with 10% ethylene glycol adipate on 45-60 mesh Chromosorb A (AW) support. Operating temperature was 190°C with a He flow rate of 300 ml/min. An Aerograph Model 1525 automatic fraction collector was connected to the gas chromatograph. Analytical
methods
The collected methyl esters were tested for purity by gas-liquid chromatography, and the quantity was determined with methyl arachidate as an internal standard. Each ester was divided equally into two portions. One portion was employed for radioactivity determination; the other portion was de-
414
carboxylated by the Schmidt reaction as described by Brady et al. [ 141, and the CO* was trapped into NCS-solubilizer (Amersham-Searle Corp., Toronto, Ontario). Tr-ens double bonds of fatty acids were identified by infrared absorption spectrometry. The geometrical isomers of the monoenoic acid were determined quantitatively by the method of Emken [15] and were analyzed by gas-liquid chromatography with a stainless steel column (270 cm X 0.2 cm), packed with 3% EGSP-Z on loo-120 Chromosorb Q. Analytical gas-liquid chromatography was performed on a Varian Aerograph Model 2100 Gas Chromatograph equipped with dual columns and dual hydrogen flame ionization detectors. The stainless steel columns (150 cm X 0.2 cm) were packed with 10% ethylene glycol succinate copolymer (EGSS-X) on 100-120 mesh Chromosorb P. The instrument was programmed from 160 to 190°C at 4” per minute. A Nuclear-Chicago Mark 1 Liquid Scintillation Counter was used for all radioactivity determinations and it employed the channels ratio quench correction procedure. Results By feeding the experimental diets for 12 weeks, the growth rate of rats fed the hydrogenated coconut oil, ethyl oleate, or ethyl elaidate was depressed significantly, and accompanied by small liver weights when compared to the corn oil control animals. The actual weights of liver total lipids and fatty acid esters expressed as mg per g of tissue, however, did not differ significantly within the four dietary regimens. One half hour f&owing the intraperitoneal injection of sodium [l-’ 4 C] acetate, no significant difference was noted between the specific activities of liver lipids of corn oil or hydrogenated coconut oil-fed rats. However, ethyl oleate or ethyl elaidate-fed animals had increased labelling compared to the control group, and the truns fatty acid group was the highest of all. When the total recovered radioactivity was presented as percent of injected dose, animals fed all three dietary fats deficient in linoleic acid showed higher values than those fed the corn oil control fat. Similar observations were found in the labelling of liver fatty acid methyl esters, and again the ethyl elaidate-fed animals were highest. As shown in Table I, the incorporated radioactivities were distributed mainly in the phospholipid, triacylglycerol and free fatty acid fractions of liver lipids. In the case of the corn oil group, approximately 60% of the total radioactivity was incorporated into the phospholipid fraction. When hydrogenated coconut oil or ethyl oleate was the dietary fat the ’ 4 C label in the phospholipid was decreased, but there was a consistent increase in the triacylglycerols. This shift in the distribution of radioactivity, however, did not affect the actual content of phospholipid (Table I). When ethyl elaidate was in the diet, in addition to a highly labelled triacylglycerol fraction, radioactivities incorporated into the fractions of phospholipids, monoacylglycerols and cholesterol esters were significantly higher than with the other dietary regimens. Free cholesterol, on the other hand, was highly labelled in the corn oil-fed animals and this is in agreement with a previous report [ 161. Separations of liver methyl esters revealed that regardless of the dietary regimen, over 95% of the incorporated [I 4 C] acetate was associated with four
OF SODIUM [l- 14C] ACETATE
INTO LIVER LIPID CLASSES
34 + 4b 49 + 2a
27 t 3b
275 f 14’ 408 ? lga
rib
34 f 5b
231 +
nlYCt?lWdS
Monoacyl-
3’
336 +
oil
Phospholipids
12 f 3b 12 + 2b
21 f gb
37 * 6a
Cholesterol
Distribution of radioactivity (dpm/mg liver)
Hydrogenated coconut oil Ethyl oleate Et.hvl elaidate
corn
Dietary groups
138 f 7a 92 t 9b
89 f 5b
80 L Eb
Free fatty acids gb
222 _+10a 224 ?- 22a
217 ?- 8a
99 +
Triacylglycerols
9 I 2b 21 ?r2a
4 i Ibc
2 f O.lC
Cholesterol esters
30.3 + 0.5b 33.3 + 1.0a
32.8 + l.lab
30.0 +_1.2b
Liver phospholipids (mg/g liver)
Values are mean f S.E. for five animals. Means which show a common superscript letter are not significantly different (P < 0.05) by Duncan’s multiple range test.
INCORPORATION
TABLE I
OF LIVER
FAT,
AND
THE INCORPORATION
OF SODIUM
/I- I 4C1 ACETATE INTO
THE FATTY AClDS
:2 20 : 3 (n-9) 20 : 4 (n-6 aad e-7) 22 : 4 (n-43 22 : 5 (n-6) 22 : 6 (n-3)
18
3.4 : 0 16 : 0 16 : 1 18 : 0 18: 1 18 : 1 trans 18 : 2 cis, trims
Fatty acids
(9)
Corn ait ~-_^l.~-~
Dietary gnxzps
-
-
-
232
2 t 4”
0.4c
(dPm/lrg>
Specific activity
1.5 t 0.1 2.6 f 0.1
I.9 t 0.6 14.7 + 0.8 9.5 L 0.7
WIO)
hydrogenated cocanut Oil .~~.l--il-xI~-.
-_-_~
+ + * t
82c 59c 48b Ifb
27 ?: 7b 6 t 0.7b 2” o.t+
614 303 222 105
(dpm/U
Specific activity
1.6 k 0.1. 2.x 1 0.1
1.4 f 0.1 15.1 f 0.8 8.3 L 0.4
Trace 21.5 ?: 0.4 4.3 t 0.4 16.5 L 0.4 27,3 f. 0.5 .._
(%t
EtbyI oleate _I_...-____-.--~_-
_~-“_,~-~~_-~__c~“_-
30+ 62 3 k
7b lb 0.2b
f: g%b *- 64b ?: 2lb * 8”
0.5 * 0.1 2.0 * 0.3
x333 t 139a 745 i- 10aa 478C 63a 205 k 20a 171 4 182 3 44 k 5a 8 * .‘la 2r 0.4b -
906 479 226 54
-
Specific activity @pm/&z) Trace 19.9 + 1.2 8.4 L 0.8 10.7 i. 0.4 23.3 -f 0.7 12.1 rt:0.4 4.5 + 0.3 2.1 1. 0.1 8.3 + 0.5 4.9 + 0.3
(%)
(dIx&&)
Specific activity
1
Ethyl etaidate _-_._“-~___-“_s.“~
._...-_^ ,~.“_~
Values are mean t S.E, for five animals. Fatty acids are expressed as axea percent by gas-Iiquid chromato@;raphy. In the essential fatty acid deficient animats, 20 : 4 is a mixture of 20 : 4 (n-6) with a sma.U amount of 20 : 4 (n-7) [II, Duncan’s multiple range test was done only for values of specific activity. Means which show a ectmmon ~~~x~~p~ iettex are not ~g~f~~ntIy different (P < 0.05).
FATTY ACID CO~PaSITKON
TABLE IX
major saturated (16 : 0 and 18 : 0) and monounsaturated (16 : 1 and 18 : 1) components (Table II); among them, as expected, palmitic acid was labelled predominantly since this one is known to be the main product of de novo fatty acid biosynthesis [17]. With one exception, for dietary groups deficient in linoleate, stearic and oleic acids were more labelled than those of the control group. The relatively low specific activity of the oleic acid noted in the ethyl oleate-fed animals reflects the greater concentration of unlabelled dietary oleic, which caused a suppression in the 9-desaturation of stearic acid. When animals were raised on the ethyl elaidate diet, all four major fatty acids (16 : 0, 16 : 1, 18 : 0, 18 : 1) showed significantly higher specific activities than did those of rats fed the other dietary regimens, Arachidonic acid (20 : 4 n - 6), on the other hand, was labelled to a much greater extent in the corn oil group. Obviously, the formation of arachidonate is almost eliminated due to the lack of linoleic acid in the other three dietary groups. It is shown in Table II that those groups deficient in linoleate, produced a substantial quantity of eicosatrienoic acid (20 : 3, n - 9) [l] , but this acid contained a low amount of label compared to the arachidonic acid in the control group. These differences in the degree of labelling from [I 4 C] acetate in the 20 : 3 or 20 : 4 acids probably are due to a different enzyme-substrate affinity for their metabolic precursors in a chain elongation system, as was demonstrated with in vitro conditions [18]. Among the essential fatty acid deficient groups, rats fed elaidate had a higher specific activity in their 20 : 3 acid; this was noted also for the 16 and l&carbon acids, but to a much greater degree. A small amount of radioactivity was observed to associate with linoleic acid and the specific activity of this acid was increased in animals fed essential fatty acid deficient diets. In rats fed ethyl elaidate, both elaidic and 5cis,9transoctadecedienoic acids containing radioactivity were deposited in the liver lipids. Decarboxylation of isolated fatty acids (Table III) showed that between 12.3 and 14.7% of the radioactivity associated with palmitic acid was in the TABLE III INCORPORATION FATTY ACIDS
OF SODIUM
[l- 14C]ACETATE
INTO
THE
CARBOXYL
CARBON
OF LIVER
Values are mean +_ S.E. for five animals. They are expressed as percentage of the radioactivities of the methyl esters. Means which show a common superscript letter are not significantly different (P < 0.05) by Duncan’s multiple range test. Fatty acids
16 16 18 18 18 18 18 20 20
0 1 0 1 1 trans 2 cis, tram 2 3 (n-9) 4 (n-6 and n-7)
Dietary groups
corn
Hydrogenated
Oil
coconut
(a)
(90)
14.0 17.1 15.1 24.0
i f + f
0.5a 3.8b o.9b 3.4a
12.5 20.5 23.5 20.4
oil
f + f *
o.4b 2.4a 2.1a 1.5b
Ethyl oleate
Ethyl elaidate
(%I
(%)
14.7 21.0 18.2 17.2
-
-
-
89.5 + 1.78 82.7 f 2.9a
84.6 + 4.3ab 66.3 + 6.4b 84.4 + 1.48
78.1
* l.oa + 2.3a * l.ob f 1.3c
5
3.9”
81.9 * 3.4a 87.8 f 4.6a
12.3 11.9 15.6 16.0 37.8 67.6 87.2 42.7 80.7
r + f k * k f t k
o.4b 0.5= zob 1.4c 0.5 5.5 3.2a 2.gc 3.7a
418
terminal carboxyl carbon. Therefore, it can be concluded that this acid was formed entirely by de novo biosynthesis. The only other liver fatty acid among the four dietary groups which showed this level of labelling in the carboxyl carbon was the palmitoleic acid from the ethyl elaidate-fed rats. This suggests that in this group the 16 : 1 acid was essentially all produced by desaturation of 16 : 0 with no extra concentration of labelling in the carboxyl carbon due to elongation or exchange with labelled acetate. The very high palmitoleate value for the ethyl oleate-fed rats has no logical explanation. However, the high values for the hydrogenated coconut oil-fed rats for 16 : 1 and 18 : 0 may be due to an elongation of short chain acids (12 : 0, 14 : 0) from the dietary source. When the levels of labelling of the 16 and B-carbon saturated and monounsaturated acids are compared with those levels for the 20 : 3 and 20 : 4 acids, it is apparent that the latter had much more activity in the carboxyl carbons. This indicates that the longer chain acids were formed to a greater extent by chain elongation. In the case of 20 : 3, 81.9 and 66.3% of the total radioactivities were incorporated into the carboxyl carbons of the acid for the ethyl oleate and hydrogenated coconut oil groups, respectively. When ethyl elaidate was the dietary fat, only 42.7% of the total for 20 : 3 was recovered in the carboxyl carbon. The value suggests that in this case the trienoic acid was produced by elongation from much more uniformly * 4 C-labelled precursors. Discussion The activity of enzymes catalyzing the synthesis of long-chain fatty acids from acetate and malonyl CoA in liver is known to be modified by various physiological and nutritional states. In regard to the latter factor, linoleic acid deficiency is found to enhance the cellular capacity for de novo fatty acid biosynthesis [ 191, With hepatic microsomal preparations, Inkpen et al. [20] reported from in vitro studies that saturated fat as hydrogenated coconut oil, which was deficient in linoleic acid, stimulated the 9-desaturase activity to convert the stearate to oleate. Results in the present experiment with rats were in agreement with these reports when hydrogenated coconut oil, or ethyl elaidate were used as dietary fats. Feeding ethyl elaidate, compared with corn oil, hydrogenated coconut oil or ethyl oleate, led to a significant increase in the incorporation of [’ 4 C] acetate into liver lipids. This is in contrast to the report by Egwim and Sgoutas [21] that partially hydrogenated soybean fat containing substantial amounts of different tram fatty acids was a better depressor of acetate incorporation into fatty acids than corn oil. It was observed also in the current study that all four major endogenously formed fatty acids, palmitic, palmitoleic, stearic and oleic were much more highly labelled in the tram acid-fed animals (Table II). These results strongly suggest that in addition to the effect of a linoleate deficiency, dietary bans acid intensifies the capacity for hepatic de novo fatty acid synthesis from acetate. This was confirmed further by the relatively low radioactivities associated with the carboxyl carbon of these four highly labelled fatty acids in the ethyl elaidate group (Table III). Elaidate feeding (Table II) resulted in the incorporation of both elaidic acid and its metabolite, cis, trans-octadecadienoic acid. However, no longerchain fatty acid from isolated components was found which contained tram
419
double bonds. Increases in the rat of endogenous fatty acid biosynthesis in the livers of elaidate-fed rats would indicate that the above Pans acids can not function in a normal way like the natural cis fatty acids, or saturated fatty acids in the essential fatty acid deficient animals. Many mechanisms which control the modifications of fatty acid synthesis are not known. As reported earlier [6,7], the lowering of 20 : 3 and 20 : 4 due to the dietary truns fatty acids was observed also in the current experiment. Following the administration of [ 1 4 C] acetate, 20 : 3 in the ethyl elaidate group had a higher specific activity than that in the other essential fatty acid deficient groups (Table II). In fact, decarboxylation of the acid revealed that most of the radioactivity associated with the trienoic acid in the former group was due to a highly labelled l&carbon precursor; in contrast, in the latter groups predominant portions of labelling were found concentrated in the carboxyl carbons of the acid. This would indicate that in the ethyl elaidate-fed animals, the addition of acetate to l&carbon unsaturated fatty acid to form 20 : 3 is not so efficient as for the other dietary regimens. Also, the high ratio for the specific activity of 18 : 1 to 20 : 3 acids in the ethyl elaidate group reflects a slow conversion of 18 : 1 to 20 : 3. In in vitro studies on fatty acid desaturation with essential fatty acid deficient-rat liver microsomes, evidence has indicated that both 18 : 0 and 20 : 2 (n - 9) are readily desaturated to 18 : 1 (n - 9) and 20 : 3 (n - 9), respectively; however, the rate of desaturation of 18 : 1 (n - 9) to 18 : 2 (n - 9) was extremely slow [22,23]. Similar observations were found in vivo with essential fatty acid deficient rats using ’ 4 C-labelled compounds [ 241. It would thus appear that with our present experimental conditions involving an essential fatty acid deficiency, the slow rate of 6-desaturation of oleic acid may be a major controlling step for the lowering of 20 : 3 acid due to the feeding of trans acid. Radioactivities incorporated into linoleic, elaidic and 5cis,9trans-octadecadienoic acids (Table II) will be given special consideration. Previous studies reported radioactivity in the 18 : 2 fraction, resulting from either exchange of [’ 4C]acetate, or the formation of isomers such as 8,11- and 7,10-octadecadienoic acids [ 25,261. In the present experiment, liver fatty acids were separated by argentation thin-layer chromatography. In the case of bans monoenoic ester, the thin-layer chromatography fraction containing it was separated clearly from saturated, or cis monounsaturated esters. A furyher purification by preparative gas-liquid chromatography produced over 99% pure fatty ester. Therefore, the radioactivity associated with this acid was due to an actual incorporation rather than any contamination. When the above three acids (18 : 2, trans-18 : 1, and cis,trans-18 : 2) were decarboxylated (Table III), this revealed that an average of 84.8% of the ’ 4 C in the 18 : 2 fraction from all dietary groups was associated with the carboxyl carbon. It appears that the predominant portion of the radioactivity in this case was due to an exchange of [I 4 C] acetate. However, radioactivities incorporated into the carboxyl carbons of cis,trans-18 : 2 and trans-18 : 1 were decreased to 67.6 and 37.8%, respectively. It appears that a smaller proportion of these two acids was affected by [’ 4 C] acetate exchange. The results from the present study indicate that dietary elaidic acid stimulated the rate of hepatic de novo fatty acid biosynthesis, which was revealed by
420
a high level of radioactivity in all four major saturated and monounsaturated fatty acids. The slow conversion of oleic acid to eicosatrienoic acid in the essential fatty acid deficient, truns acid-fed rats suggests that the 6-desaturase for monounsaturated fatty acids may be the enzyme most responsible for the decrease in the amount of long-chain unsaturated fatty acids found in these animals. Acknowledgements The authors wish to thank Dr B.L. Walker for his valuable advice and assistance. The excellent technical assistance of Miss A. Wilson was appreciated. This work was supported by the Ontario Ministry of Agriculture and Food, and the National Research Council of Canada. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Fulco. A.J. and Mead, J.F. (1959) J. Biol. Chem. 234. 1411 Klenk, E. and O&e, K. (1960) Z. Physiol. Chem. 318, 86 Privett, 0.8 and Blank, M.L. (1964) J. Am. Oil Chem. Sot. 41, 292 Selinger, 2. and Holman, R.T. (1965) Biochim. Biophys. Acta 106, 56 Privett, O.S., Stearns, Jr, E.M. and NickelI. E.C. (1967) J. Nutr. 92. 303 Lemarchal, P. and Munsch. N. (1965) Comptes Rendus 260, 714 Egwim, P.O. and Sgoutas, D.S. (1971) J. Nutr. 101. 307 Lemarchal, P. (1966) Comptes Rendus 262. 816 Dhopeshwarkar. G.A. and Mead, J.F. (1962) J. Lipid Res. 3, 238 Litchfield, C., Hariow, R.D.. Isbell, A.F. and Reiser. R. (1965) J. Am. Oil Chem. Sot. 42, 73 Folch, J., Lees, M. and Sloane Stanley, G.H. (1957) J. Biol. Chem. 226, 497 Bartlett, G.R. (1959) J. Biol. Chem. 234, 466 Morrison, W.R. and Smith, L.M. (1964) J. Lipid Res. 5, 600 Brady, R.O.. Bradley, R.M. and Trams, E.C. (1960) J. Biol. Chem. 235, 3093 Emken, E.A. (1971) Lipids 6, 686 Serdarevich, B. and Carroll, K.K. (1972) Can. J. Biochem. 50, 557 Burton, D.N., Ha&k, A.G. and Porter, J.W. (1968) Arch. Biochem. Biophys. 126. 141 Christiansen, K., Marcel, Y. and Gan, M.V. (1968) J. Biol. Chem. 243. 2969 Allmann. D.W. and Gibson, D.M. (1965) J. Lipid Res. 6, 51 Inkpen, CA.. Harris, R.A. and Quackenbush. F.W. (1969) J. Lipid Res. 10, 277 Egwin, P.O. and Sgoutas, D.S. (1972) Am. J. Clin. Nutr. 25.16 Brenner. R.R. and Peluffo, R.O. (1966) J. Biol. Chem. 241. 5213 Ullman, D. and Sprecher. H. (1971) Biochim. Biophys. Acta 248. 61 Sprecher. H.W. (1972) Fed. Proc. 31, 1451 Fulco. A.J. and Mead. J.F. (1960) J. Biol. Chem. 235, 3379 Dhopeshwarkar, G.A.. Maier, R. and Mead, J.F. (1969) Biochim. Biophys. Acta 187, 6
Biochimica 0 Elsevier
et Biophysics
Scientific
Acta,
Publishing
369
(1974)
Company,
41 l-420
Amsterdam
- Printed
in The Netherlands
BBA 56513
EFFECT OF GEOMETRIC ISOMERS OF A’-OCTADECENOIC ACID ON LIVER LIPID BIOSYNTHESIS BY ESSENTIAL FATTY ACID DEFICIENT RATS L.S.S. GUO* and J.C. ALEXANDER Department
(Received
of Nutrition,
University
of Guelph,
Guelph,
Ontario
NlG
2Wl
(Canada)
May 9th, 1974)
Summary 1. Liver lipid biosynthesis was studied by measuring the incorporation of radioactivity 0.5 h after the administration of [l-i 4 C] acetate to rats. The animals were fed 10% fat diets containing corn oil, hydrogenated coconut oil, ethyl oleate, or ethyl elaidate. 2. Incorporation of labelled acetate into liver lipids was enhanced in the three linoleic acid deficient dietary groups (hydrogenated coconut oil, ethyl oleate and ethyl elaidate). The highest level of labelling was found when ethyl elaidate was the dietary fat. 3. Patterns of distribution of ’ 4 C among liver lipid classes were altered in the hydrogenated coconut oil and ethyl oleate dietary groups compared to the corn oil control group; the label was increased in the triacylglycerols, and decreased in the phospholipids. In the case of the ethyl elaidate group, in addition to the triacylglycerols, high labelling was shown in the phospholipids, the monoacylglycerols and the cholesterol esters. 4. In the animals fed the trans fatty acid, there was a significant increase in the radioactivity in liver palmitic, palmitoleic, steak, and oleic acids. Small concentrations of these radioactivities associated with the carboxyl carbons of the acids indicate that they are formed preferentially by de novo biosynthesis. 5. A relatively small increase in the specific activity of 20 : 3 compared to 18 : 1 in the truns acid-fed group reflects a slow conversion of 18 : 1 to 20 : 3. When the radioactivity in the carboxyl carbons of the 20 : 3 acid from the rats in each of the essential fatty acid deficient groups was compared, the lowest level was found with animals fed ethyl elaidate.
* Present 94120.
address: U.S.A.
Department
of
Nutritional
Science,
University
of
California.
Berkeley,
Calif.
412
Introduction It is well established that when animals are maintained on a diet deficient in essential fatty acid, levels of 5,8,11-eicosatrienoic acid and 4,7,10,13-eicosatetraenoic acid derived from ofeic acid and palmitoleic acid are elevated in tissue lipids [ 1,2]. These biosynthetic pathways, however, have been reported to be inhibited as a result of feeding trans fatty acids [3-61. When rats were fed a partially hydrogenated soybean fat containing trans fatty acids Egwim and Sgoutas [ 71 found that the metabolites from oleate and palmitoleate were accumulated as eicosadienoic acids in the liver lipids, with corresponding low levels of eicosatrienoic and eicosatetraenoic acids due to essential fatty acid deficiency. They suggested that the occurrence of these dienoic acids was due to changes in the activity of long-chain acyl desaturases in the liver microsomes. Evidence indicates that trans-octadecenoic acid can be desaturated in vivo to a 5c~s,9trans-o~tadecadienoic acid f6,8]. On the other hand, this trans monoene also is converted to stearic acid by a direct hydrogenation process [9]. In unpublished data from our laboratory, it was found that when rats were fed a 10% elaidate diet, in addition to the deposition of elaidic and 5cis,9transoctadecadienoic acids, a substantial amount of cis monounsaturated fatty acids was observed in liver phospholipids. It appeared that this natural cis tissue acid was being formed by (1) the endogenous biosynthesis from acetate, (2) the hydrogenation of dietary elaidic acid followed by desaturation, or (3) a combination of 1 and 2. The present experiment was designed to study the first of the above synthetic routes with incorporation of [l-’ 4 C] acetate into liver lipids by rats fed either cis or tram isomers of octadecenoic acid. Since tram fatty acids have been shown in many of their biological properties to be similar to saturated fatty acids, hydrogenated coconut oil was included in addition to corn oil as a control fat. Materials and Methods
Male weanling rats of the Wistar strain, obtained Guelph, Ontario, weighing 50-56 g were used.
from Woodlyn
Farms,
Preparation of ethyl esters For this study, the ethyl oleate was isolated and esterified from total fatty acids of olive oil by Iow temperature c~stallization process, followed by a urea treatment. The final material was 98% oleate ester with a small component of palmitoleic acid. Ethyl elaidate was prepared by isomerizing oleic acid with nitrous acid [lo] . One recrystallization from light petroleum produced 90% of the tram isomer. ~x~er~~entai procedure Male weanling rats were randomly distributed into four groups of 5 animals each, and maintained on diets containing: fat (lo%), dextrose (58X%), vitamin-free casein (ZO%), DL-methionine (0.2%), cellulose (5%), salt mix,
413
U.S.P. XIV (5%) and a vitamin mix (1%). The vitamin mixture contained the following per kilogram of diet: (in I.U.) vitamin A, 24 000; vitamin D, 4000; (in mg) vitamin E (as cw-tocopherol), 120: menadione, 1.0; thiamine - HCl, 10; pyridoxine * HCl, 5; niacin, 50; calcium pantothenate, 20; choline chloride, 1000; p-aminobenzoic acid, 20; inositol, 1000; riboflavin, 10; folic acid, 1.0; biotin, 0.5; (in pg) vitamin B1 *, 10. Corn oil, hydrogenated coconut oil, ethyl oleate and ethyl elaidate each served as a fat source in different dietary groups. The rats were fed the experimental diets for 12 weeks. During the last two weeks, the animals were mealfed only 2 h daily. Soon after the end of the feeding period on the last day of Corp., the experiment 25 /.K!i of sodium [l-l 4 C] acetate ( Amersham-Searle Toronto, Ontario) per 100 g of body weight was given to each rat by intraperitoneal injection. A preliminary experiment showed that the rate of incorporation of [’ 4 C] acetate into liver lipids of both corn oil and hydrogenated coconut oil-prefed rats was parallel within a 4 h interval following the injection of labelled compound. The peak of the incorporation was at 0.5 h after the administration. Therefore, this time period was selected for tissue collection. The animals were exsanguinated under diethyl ether anesthesia, their livers were removed, weighed and immediately homogenized. Lipid extraction
and fractionation
Lipids were extracted from the homogenized tissues three times with chloroform-methanol (2 : 1, v/v) by the procedure of Folch et al. [ 111. All combined extracts were analyzed for phospholipid content by the method of Bartlett [12]. A portion of total lipids was separated into different classes by thin-layer chromatography on Silica gel G. The plates were developed with light petroleum-diethyl ether-acetic acid (90 : 10 : 1, by vol.). Components were scraped from the plates and extracted three times into scintillation vials. For the extraction of the components of neutral lipids, diethyl ether was used, while the phospholipid fraction was extracted with methanol. The remaining portion of the lipids which was not used for thin-layer chromatography was interesterified with 14% borontrifluoride-methanol (w/v) under N 2 [ 131 and the methyl esters were separated from the free cholesterol by a silicic acid column. These fatty acid esters were fractionated by argentation thin-layer chromatography according to their degrees of unsaturation. The methyl ester fractions were further separated or purified by preparative gasliquid chromatography. An Aerograph Model 1520 Gas Chromatograph equipped with a thermal conductivity detector, and coiled aluminum columns (360 cm X 0.6 cm) was used. The columns were packed with 10% ethylene glycol adipate on 45-60 mesh Chromosorb A (AW) support. Operating temperature was 190°C with a He flow rate of 300 ml/min. An Aerograph Model 1525 automatic fraction collector was connected to the gas chromatograph. Analytical
methods
The collected methyl esters were tested for purity by gas-liquid chromatography, and the quantity was determined with methyl arachidate as an internal standard. Each ester was divided equally into two portions. One portion was employed for radioactivity determination; the other portion was de-
414
carboxylated by the Schmidt reaction as described by Brady et al. [ 141, and the CO* was trapped into NCS-solubilizer (Amersham-Searle Corp., Toronto, Ontario). Tr-ens double bonds of fatty acids were identified by infrared absorption spectrometry. The geometrical isomers of the monoenoic acid were determined quantitatively by the method of Emken [15] and were analyzed by gas-liquid chromatography with a stainless steel column (270 cm X 0.2 cm), packed with 3% EGSP-Z on loo-120 Chromosorb Q. Analytical gas-liquid chromatography was performed on a Varian Aerograph Model 2100 Gas Chromatograph equipped with dual columns and dual hydrogen flame ionization detectors. The stainless steel columns (150 cm X 0.2 cm) were packed with 10% ethylene glycol succinate copolymer (EGSS-X) on 100-120 mesh Chromosorb P. The instrument was programmed from 160 to 190°C at 4” per minute. A Nuclear-Chicago Mark 1 Liquid Scintillation Counter was used for all radioactivity determinations and it employed the channels ratio quench correction procedure. Results By feeding the experimental diets for 12 weeks, the growth rate of rats fed the hydrogenated coconut oil, ethyl oleate, or ethyl elaidate was depressed significantly, and accompanied by small liver weights when compared to the corn oil control animals. The actual weights of liver total lipids and fatty acid esters expressed as mg per g of tissue, however, did not differ significantly within the four dietary regimens. One half hour f&owing the intraperitoneal injection of sodium [l-’ 4 C] acetate, no significant difference was noted between the specific activities of liver lipids of corn oil or hydrogenated coconut oil-fed rats. However, ethyl oleate or ethyl elaidate-fed animals had increased labelling compared to the control group, and the truns fatty acid group was the highest of all. When the total recovered radioactivity was presented as percent of injected dose, animals fed all three dietary fats deficient in linoleic acid showed higher values than those fed the corn oil control fat. Similar observations were found in the labelling of liver fatty acid methyl esters, and again the ethyl elaidate-fed animals were highest. As shown in Table I, the incorporated radioactivities were distributed mainly in the phospholipid, triacylglycerol and free fatty acid fractions of liver lipids. In the case of the corn oil group, approximately 60% of the total radioactivity was incorporated into the phospholipid fraction. When hydrogenated coconut oil or ethyl oleate was the dietary fat the ’ 4 C label in the phospholipid was decreased, but there was a consistent increase in the triacylglycerols. This shift in the distribution of radioactivity, however, did not affect the actual content of phospholipid (Table I). When ethyl elaidate was in the diet, in addition to a highly labelled triacylglycerol fraction, radioactivities incorporated into the fractions of phospholipids, monoacylglycerols and cholesterol esters were significantly higher than with the other dietary regimens. Free cholesterol, on the other hand, was highly labelled in the corn oil-fed animals and this is in agreement with a previous report [ 161. Separations of liver methyl esters revealed that regardless of the dietary regimen, over 95% of the incorporated [I 4 C] acetate was associated with four
OF SODIUM [l- 14C] ACETATE
INTO LIVER LIPID CLASSES
34 + 4b 49 + 2a
27 t 3b
275 f 14’ 408 ? lga
rib
34 f 5b
231 +
nlYCt?lWdS
Monoacyl-
3’
336 +
oil
Phospholipids
12 f 3b 12 + 2b
21 f gb
37 * 6a
Cholesterol
Distribution of radioactivity (dpm/mg liver)
Hydrogenated coconut oil Ethyl oleate Et.hvl elaidate
corn
Dietary groups
138 f 7a 92 t 9b
89 f 5b
80 L Eb
Free fatty acids gb
222 _+10a 224 ?- 22a
217 ?- 8a
99 +
Triacylglycerols
9 I 2b 21 ?r2a
4 i Ibc
2 f O.lC
Cholesterol esters
30.3 + 0.5b 33.3 + 1.0a
32.8 + l.lab
30.0 +_1.2b
Liver phospholipids (mg/g liver)
Values are mean f S.E. for five animals. Means which show a common superscript letter are not significantly different (P < 0.05) by Duncan’s multiple range test.
INCORPORATION
TABLE I
OF LIVER
FAT,
AND
THE INCORPORATION
OF SODIUM
/I- I 4C1 ACETATE INTO
THE FATTY AClDS
:2 20 : 3 (n-9) 20 : 4 (n-6 aad e-7) 22 : 4 (n-43 22 : 5 (n-6) 22 : 6 (n-3)
18
3.4 : 0 16 : 0 16 : 1 18 : 0 18: 1 18 : 1 trans 18 : 2 cis, trims
Fatty acids
(9)
Corn ait ~-_^l.~-~
Dietary gnxzps
-
-
-
232
2 t 4”
0.4c
(dPm/lrg>
Specific activity
1.5 t 0.1 2.6 f 0.1
I.9 t 0.6 14.7 + 0.8 9.5 L 0.7
WIO)
hydrogenated cocanut Oil .~~.l--il-xI~-.
-_-_~
+ + * t
82c 59c 48b Ifb
27 ?: 7b 6 t 0.7b 2” o.t+
614 303 222 105
(dpm/U
Specific activity
1.6 k 0.1. 2.x 1 0.1
1.4 f 0.1 15.1 f 0.8 8.3 L 0.4
Trace 21.5 ?: 0.4 4.3 t 0.4 16.5 L 0.4 27,3 f. 0.5 .._
(%t
EtbyI oleate _I_...-____-.--~_-
_~-“_,~-~~_-~__c~“_-
30+ 62 3 k
7b lb 0.2b
f: g%b *- 64b ?: 2lb * 8”
0.5 * 0.1 2.0 * 0.3
x333 t 139a 745 i- 10aa 478C 63a 205 k 20a 171 4 182 3 44 k 5a 8 * .‘la 2r 0.4b -
906 479 226 54
-
Specific activity @pm/&z) Trace 19.9 + 1.2 8.4 L 0.8 10.7 i. 0.4 23.3 -f 0.7 12.1 rt:0.4 4.5 + 0.3 2.1 1. 0.1 8.3 + 0.5 4.9 + 0.3
(%)
(dIx&&)
Specific activity
1
Ethyl etaidate _-_._“-~___-“_s.“~
._...-_^ ,~.“_~
Values are mean t S.E, for five animals. Fatty acids are expressed as axea percent by gas-Iiquid chromato@;raphy. In the essential fatty acid deficient animats, 20 : 4 is a mixture of 20 : 4 (n-6) with a sma.U amount of 20 : 4 (n-7) [II, Duncan’s multiple range test was done only for values of specific activity. Means which show a ectmmon ~~~x~~p~ iettex are not ~g~f~~ntIy different (P < 0.05).
FATTY ACID CO~PaSITKON
TABLE IX
major saturated (16 : 0 and 18 : 0) and monounsaturated (16 : 1 and 18 : 1) components (Table II); among them, as expected, palmitic acid was labelled predominantly since this one is known to be the main product of de novo fatty acid biosynthesis [17]. With one exception, for dietary groups deficient in linoleate, stearic and oleic acids were more labelled than those of the control group. The relatively low specific activity of the oleic acid noted in the ethyl oleate-fed animals reflects the greater concentration of unlabelled dietary oleic, which caused a suppression in the 9-desaturation of stearic acid. When animals were raised on the ethyl elaidate diet, all four major fatty acids (16 : 0, 16 : 1, 18 : 0, 18 : 1) showed significantly higher specific activities than did those of rats fed the other dietary regimens, Arachidonic acid (20 : 4 n - 6), on the other hand, was labelled to a much greater extent in the corn oil group. Obviously, the formation of arachidonate is almost eliminated due to the lack of linoleic acid in the other three dietary groups. It is shown in Table II that those groups deficient in linoleate, produced a substantial quantity of eicosatrienoic acid (20 : 3, n - 9) [l] , but this acid contained a low amount of label compared to the arachidonic acid in the control group. These differences in the degree of labelling from [I 4 C] acetate in the 20 : 3 or 20 : 4 acids probably are due to a different enzyme-substrate affinity for their metabolic precursors in a chain elongation system, as was demonstrated with in vitro conditions [18]. Among the essential fatty acid deficient groups, rats fed elaidate had a higher specific activity in their 20 : 3 acid; this was noted also for the 16 and l&carbon acids, but to a much greater degree. A small amount of radioactivity was observed to associate with linoleic acid and the specific activity of this acid was increased in animals fed essential fatty acid deficient diets. In rats fed ethyl elaidate, both elaidic and 5cis,9transoctadecedienoic acids containing radioactivity were deposited in the liver lipids. Decarboxylation of isolated fatty acids (Table III) showed that between 12.3 and 14.7% of the radioactivity associated with palmitic acid was in the TABLE III INCORPORATION FATTY ACIDS
OF SODIUM
[l- 14C]ACETATE
INTO
THE
CARBOXYL
CARBON
OF LIVER
Values are mean +_ S.E. for five animals. They are expressed as percentage of the radioactivities of the methyl esters. Means which show a common superscript letter are not significantly different (P < 0.05) by Duncan’s multiple range test. Fatty acids
16 16 18 18 18 18 18 20 20
0 1 0 1 1 trans 2 cis, tram 2 3 (n-9) 4 (n-6 and n-7)
Dietary groups
corn
Hydrogenated
Oil
coconut
(a)
(90)
14.0 17.1 15.1 24.0
i f + f
0.5a 3.8b o.9b 3.4a
12.5 20.5 23.5 20.4
oil
f + f *
o.4b 2.4a 2.1a 1.5b
Ethyl oleate
Ethyl elaidate
(%I
(%)
14.7 21.0 18.2 17.2
-
-
-
89.5 + 1.78 82.7 f 2.9a
84.6 + 4.3ab 66.3 + 6.4b 84.4 + 1.48
78.1
* l.oa + 2.3a * l.ob f 1.3c
5
3.9”
81.9 * 3.4a 87.8 f 4.6a
12.3 11.9 15.6 16.0 37.8 67.6 87.2 42.7 80.7
r + f k * k f t k
o.4b 0.5= zob 1.4c 0.5 5.5 3.2a 2.gc 3.7a
418
terminal carboxyl carbon. Therefore, it can be concluded that this acid was formed entirely by de novo biosynthesis. The only other liver fatty acid among the four dietary groups which showed this level of labelling in the carboxyl carbon was the palmitoleic acid from the ethyl elaidate-fed rats. This suggests that in this group the 16 : 1 acid was essentially all produced by desaturation of 16 : 0 with no extra concentration of labelling in the carboxyl carbon due to elongation or exchange with labelled acetate. The very high palmitoleate value for the ethyl oleate-fed rats has no logical explanation. However, the high values for the hydrogenated coconut oil-fed rats for 16 : 1 and 18 : 0 may be due to an elongation of short chain acids (12 : 0, 14 : 0) from the dietary source. When the levels of labelling of the 16 and B-carbon saturated and monounsaturated acids are compared with those levels for the 20 : 3 and 20 : 4 acids, it is apparent that the latter had much more activity in the carboxyl carbons. This indicates that the longer chain acids were formed to a greater extent by chain elongation. In the case of 20 : 3, 81.9 and 66.3% of the total radioactivities were incorporated into the carboxyl carbons of the acid for the ethyl oleate and hydrogenated coconut oil groups, respectively. When ethyl elaidate was the dietary fat, only 42.7% of the total for 20 : 3 was recovered in the carboxyl carbon. The value suggests that in this case the trienoic acid was produced by elongation from much more uniformly * 4 C-labelled precursors. Discussion The activity of enzymes catalyzing the synthesis of long-chain fatty acids from acetate and malonyl CoA in liver is known to be modified by various physiological and nutritional states. In regard to the latter factor, linoleic acid deficiency is found to enhance the cellular capacity for de novo fatty acid biosynthesis [ 191, With hepatic microsomal preparations, Inkpen et al. [20] reported from in vitro studies that saturated fat as hydrogenated coconut oil, which was deficient in linoleic acid, stimulated the 9-desaturase activity to convert the stearate to oleate. Results in the present experiment with rats were in agreement with these reports when hydrogenated coconut oil, or ethyl elaidate were used as dietary fats. Feeding ethyl elaidate, compared with corn oil, hydrogenated coconut oil or ethyl oleate, led to a significant increase in the incorporation of [’ 4 C] acetate into liver lipids. This is in contrast to the report by Egwim and Sgoutas [21] that partially hydrogenated soybean fat containing substantial amounts of different tram fatty acids was a better depressor of acetate incorporation into fatty acids than corn oil. It was observed also in the current study that all four major endogenously formed fatty acids, palmitic, palmitoleic, stearic and oleic were much more highly labelled in the tram acid-fed animals (Table II). These results strongly suggest that in addition to the effect of a linoleate deficiency, dietary bans acid intensifies the capacity for hepatic de novo fatty acid synthesis from acetate. This was confirmed further by the relatively low radioactivities associated with the carboxyl carbon of these four highly labelled fatty acids in the ethyl elaidate group (Table III). Elaidate feeding (Table II) resulted in the incorporation of both elaidic acid and its metabolite, cis, trans-octadecadienoic acid. However, no longerchain fatty acid from isolated components was found which contained tram
419
double bonds. Increases in the rat of endogenous fatty acid biosynthesis in the livers of elaidate-fed rats would indicate that the above Pans acids can not function in a normal way like the natural cis fatty acids, or saturated fatty acids in the essential fatty acid deficient animals. Many mechanisms which control the modifications of fatty acid synthesis are not known. As reported earlier [6,7], the lowering of 20 : 3 and 20 : 4 due to the dietary truns fatty acids was observed also in the current experiment. Following the administration of [ 1 4 C] acetate, 20 : 3 in the ethyl elaidate group had a higher specific activity than that in the other essential fatty acid deficient groups (Table II). In fact, decarboxylation of the acid revealed that most of the radioactivity associated with the trienoic acid in the former group was due to a highly labelled l&carbon precursor; in contrast, in the latter groups predominant portions of labelling were found concentrated in the carboxyl carbons of the acid. This would indicate that in the ethyl elaidate-fed animals, the addition of acetate to l&carbon unsaturated fatty acid to form 20 : 3 is not so efficient as for the other dietary regimens. Also, the high ratio for the specific activity of 18 : 1 to 20 : 3 acids in the ethyl elaidate group reflects a slow conversion of 18 : 1 to 20 : 3. In in vitro studies on fatty acid desaturation with essential fatty acid deficient-rat liver microsomes, evidence has indicated that both 18 : 0 and 20 : 2 (n - 9) are readily desaturated to 18 : 1 (n - 9) and 20 : 3 (n - 9), respectively; however, the rate of desaturation of 18 : 1 (n - 9) to 18 : 2 (n - 9) was extremely slow [22,23]. Similar observations were found in vivo with essential fatty acid deficient rats using ’ 4 C-labelled compounds [ 241. It would thus appear that with our present experimental conditions involving an essential fatty acid deficiency, the slow rate of 6-desaturation of oleic acid may be a major controlling step for the lowering of 20 : 3 acid due to the feeding of trans acid. Radioactivities incorporated into linoleic, elaidic and 5cis,9trans-octadecadienoic acids (Table II) will be given special consideration. Previous studies reported radioactivity in the 18 : 2 fraction, resulting from either exchange of [’ 4C]acetate, or the formation of isomers such as 8,11- and 7,10-octadecadienoic acids [ 25,261. In the present experiment, liver fatty acids were separated by argentation thin-layer chromatography. In the case of bans monoenoic ester, the thin-layer chromatography fraction containing it was separated clearly from saturated, or cis monounsaturated esters. A furyher purification by preparative gas-liquid chromatography produced over 99% pure fatty ester. Therefore, the radioactivity associated with this acid was due to an actual incorporation rather than any contamination. When the above three acids (18 : 2, trans-18 : 1, and cis,trans-18 : 2) were decarboxylated (Table III), this revealed that an average of 84.8% of the ’ 4 C in the 18 : 2 fraction from all dietary groups was associated with the carboxyl carbon. It appears that the predominant portion of the radioactivity in this case was due to an exchange of [I 4 C] acetate. However, radioactivities incorporated into the carboxyl carbons of cis,trans-18 : 2 and trans-18 : 1 were decreased to 67.6 and 37.8%, respectively. It appears that a smaller proportion of these two acids was affected by [’ 4 C] acetate exchange. The results from the present study indicate that dietary elaidic acid stimulated the rate of hepatic de novo fatty acid biosynthesis, which was revealed by
420
a high level of radioactivity in all four major saturated and monounsaturated fatty acids. The slow conversion of oleic acid to eicosatrienoic acid in the essential fatty acid deficient, truns acid-fed rats suggests that the 6-desaturase for monounsaturated fatty acids may be the enzyme most responsible for the decrease in the amount of long-chain unsaturated fatty acids found in these animals. Acknowledgements The authors wish to thank Dr B.L. Walker for his valuable advice and assistance. The excellent technical assistance of Miss A. Wilson was appreciated. This work was supported by the Ontario Ministry of Agriculture and Food, and the National Research Council of Canada. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Fulco. A.J. and Mead, J.F. (1959) J. Biol. Chem. 234. 1411 Klenk, E. and O&e, K. (1960) Z. Physiol. Chem. 318, 86 Privett, 0.8 and Blank, M.L. (1964) J. Am. Oil Chem. Sot. 41, 292 Selinger, 2. and Holman, R.T. (1965) Biochim. Biophys. Acta 106, 56 Privett, O.S., Stearns, Jr, E.M. and NickelI. E.C. (1967) J. Nutr. 92. 303 Lemarchal, P. and Munsch. N. (1965) Comptes Rendus 260, 714 Egwim, P.O. and Sgoutas, D.S. (1971) J. Nutr. 101. 307 Lemarchal, P. (1966) Comptes Rendus 262. 816 Dhopeshwarkar. G.A. and Mead, J.F. (1962) J. Lipid Res. 3, 238 Litchfield, C., Hariow, R.D.. Isbell, A.F. and Reiser. R. (1965) J. Am. Oil Chem. Sot. 42, 73 Folch, J., Lees, M. and Sloane Stanley, G.H. (1957) J. Biol. Chem. 226, 497 Bartlett, G.R. (1959) J. Biol. Chem. 234, 466 Morrison, W.R. and Smith, L.M. (1964) J. Lipid Res. 5, 600 Brady, R.O.. Bradley, R.M. and Trams, E.C. (1960) J. Biol. Chem. 235, 3093 Emken, E.A. (1971) Lipids 6, 686 Serdarevich, B. and Carroll, K.K. (1972) Can. J. Biochem. 50, 557 Burton, D.N., Ha&k, A.G. and Porter, J.W. (1968) Arch. Biochem. Biophys. 126. 141 Christiansen, K., Marcel, Y. and Gan, M.V. (1968) J. Biol. Chem. 243. 2969 Allmann. D.W. and Gibson, D.M. (1965) J. Lipid Res. 6, 51 Inkpen, CA.. Harris, R.A. and Quackenbush. F.W. (1969) J. Lipid Res. 10, 277 Egwin, P.O. and Sgoutas, D.S. (1972) Am. J. Clin. Nutr. 25.16 Brenner. R.R. and Peluffo, R.O. (1966) J. Biol. Chem. 241. 5213 Ullman, D. and Sprecher. H. (1971) Biochim. Biophys. Acta 248. 61 Sprecher. H.W. (1972) Fed. Proc. 31, 1451 Fulco. A.J. and Mead. J.F. (1960) J. Biol. Chem. 235, 3379 Dhopeshwarkar, G.A.. Maier, R. and Mead, J.F. (1969) Biochim. Biophys. Acta 187, 6