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The fatty acids of individual liver phosphatides from rats grown on a fat-free diet
BIOCHIMICA ET BIOPHYSICA ACTA
502 BBA 55107 THE FATTY ACIDS OF INDIVIDUAL GROWN
OX A FAT-FREE
LIVER PHOSPHATIDES
FROM RATS
DIET*
KONRAD TISCHER AND JOSEPH L. GLENN** Department (Received
of Biochemistry,
Albany
Medical College of Union University, Albany,
N. Y. (U.S.A.)
August 10th. 1964)
SUMMARY I. The purpose of the present study was to analyze the fatty acid composition of individual liver phosphatides from rats fed a fat-free diet for a r2-week period and commencing at the time of weaning. The effects of supplementation of either linoleic acid or linolenic acid to such a diet were also recorded. 2. The level of 5,8,rr-eicosatrienoic acid rose sharply in all phosphatides as a result of the fat-free diet, with the exception of cardiolipin, where large increases in oleic acid and palmitoleic acid were observed. 3. Daily supplementation of either linoleic acid or linolenic acid were effective in suppressing the synthesis of 5,8,x1-eicosatrienoic acid. The administration of linoleic acid with the fat-free diet resulted in the appearance of docosatetraenoic acid in some of the phosphatides, while linolenic acid supplementation led to the occurrence of eicosapentaenoic acid in all of the phosphatides.
INTRODUCTION It has been known for some time that rats, when reared on a fat-free diet, exhibited fatty acid patterns in their tissues which were different from the tissue fatty acids from rats that were grown on a normal stock diet. The earlier work of BURR AND BURR’ demonstrated that the symptoms of fat deficiency in the rat could be corrected by supplementation with fatty acids derived from lard. Recent reviews8Bphave discussed the dietary importance of both linoleic acid and linolenic acid, and a recent study by MOHBBAUERAND HOLMAN~illustrated how the dietary level of essential fatty acids affected the fatty acid composition of the total lipid fraction of rat liver. This study, although of considerable significance, did not permit the analyses of the fatty acids in the separate components of liver lipids. The primary intent of the present study was to analyze the fatty acid pattern of the individual phosphatides from rats, which were reared on a synthetic fat-free l Abbreviations: FF, rats fed with a fat free test diet; FFL, rats fed with a fat free test diet plus daily doses of linoleic acid methyl ester; FFLE, rats fed with a fat free test diet plus daily doses linolenic acid methyl ester. l l A preliminary report of this work has been published 1.
Biochim.
Biophys.
Acta, g8 (1965) 502-51 I
PHOSPHATIDE
FATTY ACIDS OF ANIMALS ON FAT-FREE
503
DIET
diet from the time of weaning. In addition, the effects of daily supplementation of either linoleic acid or linolenic acid were also studied. The results of this study demonstrated the molecular changes that occurred in the liver phosphatides of rats on a fatfree diet and can be compared to the previously known changes that occurred on the tissue level. MATERIALS
AND METHODS
Animals All of the animals used in this study were female Wistar rats obtained from the Blue Spruce Animal Farms, Altamont, New York. The weights of these animals at the time of weaning were between 40-50 g. Diet and chemicals The basal diet was the Fat-Free Test Diet purchased from the Nutritional Biochemical Corporation, Cleveland, Ohio. Exhaustive extraction of samples of this diet with chloroform-methanol (z:I, v/v) demonstrated that the diet contained minute and insignificant traces of fatty acids. Linoleic acid methyl ester and linolenic acid methyl ester were obtained from Mann Chemical Research Company, New York, N.Y. Silicic acid and organic solvents of Analytical Reagent Grade were purchased from the Mallinckrodt Chemical Works, St. Louis, MO. The fatty acid methyl ester standards employed in gas-liquid chromatography were generously donated by the National Institutes of Health, Bethesda, hid., and Applied Science Labs, State College, Pa. All analyses of fatty acids were performed on a Barber-Coleman Model IO apparatus with a diethylene-glycol-succinate phase, coated on acid washed Chromosorb W (15.4% by weight, 80-100 mesh). The fatty acid composition of each phosphatide was determined by the triangulation method, and the values reported are as area per cent. Exfwimentd design A total of 107 female rats were used in this study. One group (FF) received only the fat-free test diet, while a second group (FFL) received the fat-free diet plus daily doses of linoleic acid methyl ester, and a third group (FFLE) received the fat-free diet plus daily supplementation of linolenic acid methyl ester. II weanling rats were used to obtain normal values and were designated as the control group. The livers from the control group were obtained immediately after decapitation and the total lipids were extracted and purified according to the method of FOLCH and co-workers?. Separation and identification of the individual phosphatides, and the methylation of the phosphatide fatty acids were performed as described previously’. Subsequently, the phosphatides were isolated from the livers of rats that were on their respective diets for I, 2, 3, 5.7, g and 12 weeks. The fatty acid pattern of each phosphatide was determined at these time intervals. At least four female rats were included at each time interval in each dietary group. 4 adult female rats, 14 weeks old, were raised on a normal stock diet and their liver phosphatide fatty acids were analyzed and served as comparative end values. The fat-free diet was available to the three dietary groups at all times. The daily Biochim.
Biophys.
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98 (1965) 502-51
I
K. TISCHER, J. L. GLENN
504
doses of linoieic acid and linolenic acid, as their methyl esters, were given orally with serological pipettes. The amount of each acid administered was 50 mg per day per rat for the iirst six weeks following weaning. Beginning with the seventh week and Iasting through the twelfth week, each rat received roe mg of the acid under study. Any of the rats which failed to eat the diet or showed signs of infection were discarded. 0.2 ml
RESULTS
The elution pattern of phosphatides off a silicic acid column is shown in Fig. I. The data illustrated were obtained from 5 g of liver which were removed from 3 weanling rats immediately after decapitation. 5 fractions were detected: viz. cardiolipin;
e TUBE NUMBER Fig. I. Fractionation of weanling rat liver phosphatides on siIicic acid column: CL, cardiolipin. PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; E’C I and PC II, initial and latter parts of phosphatidylcholine. The ratios 4/r and 312 refer to chloroform/ methanol ratios that were used to elute the phosphatides.
phosphatidylethanolamine; phosphatidylinositol; phosphatidyiserine; and phosphatidylcholine. The latter phosphatide was sub divided into 2 parts, because of previously demonstrated differences iu the fatty acid composition of the initial and latter parts of this fractions. A sixth rat liver phosphatide, sphingomyelin, could be eluted off the column with a higher concentration of methanol, but this compound was not analyzed in this investigation. The elution patterns of phosphatides from the rats on the 3 experimental diets showed little or no change from that of weanling rats, nor did the absolute amounts bmoles per g of wet liver) of the individual phosphatides vary significantly on any of the diets. There were, however, marked changes in the fatty acyl groups of the phosphatides during the 12 week periods, and these alterations were recorded by the following procedures. The 3 peak tubes of each fraction, with the exception of phosphatidylcholine, were combined and the phospholipid fatty acids that were present in these tubes were determined as their methyl esters* by gas-liquid chromatography. The phosphatidylcholine region was analyzed by detenniuiug the fatty acyl groups Biochim. Biophys.
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PHOSPHATIDE FATTY ACIDS OF ANIMALS ON FAT-FREE DIET
505
TABLE I FATTYACID COMPOSITION OF RAT LIVER PHOSPHATIDES AFTER 12 WEEK DIETARY PERIODS Com#ound Cardiolipin
Phosphatidylethanolamine
Diet*
Phosphatidylserine
Phosphatidylcholine I
Phosphatidylcholine II
16:r
18:o
18:1
18:a
4.7
2.0
78.5
30.1 11.2 14.8
1.7 2.8 2.3 5.3
13.2
6.6 5.6 9.9
44.3 29.0 40.8
7.2 38.2 3.0
22.0 12.5 16.4 17.2
trace 1.9 0.7 0.7
26.5 31.9 21.9 29.5
2.3 14.4 6.5 5.5
2.6 0.7 2.2 trace
W FF FFL FFLE
5.6 2.1
trace 5.7
45.4 53.3
2.6 6.5
5.8 0.9
3.9 3.3
0.5 2.0
45.0 46.7
2.8 6.1
W FF FFL FFLE
6.0 4.9
trace 5.1
40.2 44.7
4.1 3.5
1.3 1.5
W FF FFL FFLE
21.0 7.0 5.5 8.0
trace I.3 0.3 trace
W FF FFL FFLE W FF FFL
FFLE Phosphatidylinositol
16:o
W FF
FFL FFLE
x8:3
aor
ao:q
20:s
22:4
22:5
2a:6
-
trace -
1.1 13.5
4.0 5.9 0.7
trace 3.8 4.0 trace
trace 1.5
-
-
trace
21.0 1.6
20.8 10.7 38.7 3.2
trace 15.0
trace 10.8 -
2.0 0.7
-
1.1 29.4
38.4 2.3
-
-
-
-
1.8
39.1 6.3
5.1 10.4
5.1 trace
-
14.9
24.9 10.3
-
46.8 46.7
5.1 6.8
1.5 0.3
trace 2.6
31.1 3.1
-
8.0
-
29.3 34.3 35.6
3.8 11.6 5.1 6.6
3.8 trace I.9 -
-
34.0 1.3 3.6
21.1 6.0 35.6
trace 16.8
0.5 0.4
1.5
7.5
-
1.0
9.0 18.1
0.9
6.5 I.2 26.7
-
1.0
20.2
1.9 10.2 -
trace
4.3 3.5 21.0
-
-
0.8
15.4
8.9
15.0
-
-
15.7
-
-
6.1 2.4 3.9
23.9 21.8 24.3
26.9 13.7 23.2
trace 8.2 0.6
-
I7.7 0.8 2.0
3.9 26.0 1.6
13.5
-
8.5
2.3 -
-
* W, phosphatides were isolated from weanling rats.
in 3 tubes in the middle of the ascending slope (PC I), and in 3 tubes in the middle of the descending slope (PC II). It was decided that the sampling techniques described above, rather than the combining of all the tubes in each fraction, would yield more accurate data and also avoid any overlap between individual components. The fatty acid changes which occurred in the individual phosphatides are discussed separately in the following sections, but an overall comparison in the fatty acyl composition of phospholipids from weanling rat liver and in the livers from rats which were on their respective diets for 12 weeks is presented in Table I. Fatty acid com$osition of cardiolifiin This molecule was characterized in weanling rat liver, as well as in adult rat liver, by having a very high level of linoleic acid (78%) and appreciable amounts of oleic acid (13%). Major changes which occurred in the fatty acyl content of cardiolipin during the 12 week dietary periods, are shown in Fig. 2. The most drastic alteration in this component, when the rats were placed on the FF diet, was the precipitous fall in the level of linoleic acid which decreased to 22% after only I week on the diet. After 12 weeks, the linoleic acid content of cardiolipin was 7%. To compensate for this loss, there were large increases in the levels of oleic acid (from 13% to 44%), and of pahnitoleic acid (from 2% to 30%) at the end of 12 Biofihys.
23.3 7.0 3.5 26. I
trace 1.2
33.5
Biochim.
10.7
2.9
-
21.6 25.4 23.3
0.4 1.5
-
-
-
4.3 16.5
-
-
42.3
2.5
trace
Acta, 98 (1965) 502-511
9.6 0.4 6.8
K. TISCHER, J. L. GLENN
‘0 la
FF
I
FF (0
WEEKS ON WETS Fig. 2. Major changes in fatty acid composition periods. Symbols: o-o, linoleic acid; x-x docosahexaenoic acid.
WEEKS
ON DIETS
of cardiolipin during FF, FFL, and FFLE dietary , oleic acid; O-O, palmitoleic acid: A-A,
Fig. 3. Major changes in fatty acid composition of phosphatidylethanolamine during FF. FFL, and FFLE dietary periods, Symbols: o-0, arachidonic acid; x -x , docosahexaenoic acid; O-C, docosatetraenoic acid; A-A, eicosatrienoic acid; A---A, eicosapentaenoic acid.
weeks. There was only a small amount of eicosatrienoic acid* incorporated into cardiolipin as a consequence of the FF diet, although the livers of animals on such a diet are known to have high concentrations of this acidlo. When the rats were raised on the FFL diet, there was still a decrease in the level of linoleic acid in cardiolipin, but it was not nearly as pronounced as that seen on the FF diet. Apparently, the level of linoleic acid that was administered, 50 mg in ester form per day for the first 6 weeks, and IOO mg per day for the remaining weeks, was not sufficient to maintain a normal level of this acid in cardiolipin. The decrease observed was accompanied by elevation of oleic acid and palmitoleic acid. The third diet (FFLE), that of linolenic acid methyl ester supplementation to an otherwise fat-free diet, resulted in an almost complete disappearance of linoleic acid from cardiolipin. Concomitant with this loss, there were large increases in oleic acid and palmitoleic acid, as well as the appearance of linolenic acid, docosapentaenoic acid and docosahexaenoic acid in the molecule. The last three acids were present in trace amounts in cardiolipin from weanling and adult rat liver. Fatty acid composition of phosphatidyletha+whzmine In weanling rat liver, this phospholipid contained high levels of palmitic acid (22%), stearic acid (26%), arachidonic acid (21%) and docosahexaenoic acid (23%).
l 5,8,1x-eicosatrienoic acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexanoic acid were identified by relative retention times and‘the use of standards.
Biochim.
Biophys.
Actn, 98 (1965) 502-51 I
PHOSPHATIDE FATTY ACIDS OF ANIMALS ON FAT-FREE
DIET
507
All of these acids, with the exception of stearic acid, decreased in phosphatidylethanolamine when the animals were placed on the FF diet. However, the most interesting changes were observed in the levels of the polyunsaturated acids and they are recorded in Fig. 3. along with the alterations found in this phosphatide as a consequence of the other diets. On the FF diet, arachidonic decreased gradually while the level of docosahexaenoic acid dropped sharply and appeared to stabilize at approx. 5%. The above losses were compensated for by a marked elevation in 5,8,rr-eicosatrienoic acid, which was not present in weanling rat liver phosphatidylethanolamine, to a level of 21% after 12 weeks. Although not shown in Fig. 3, oleic acid was also elevated from 2% to 14% at the conclusion of the FF dietary period. In the FFL animals, the level of linoleic acid in phosphatidylethanolamine did not change significantly, but arachidonic acid content increased steadily to reach a level of 39% after the 12 week period. The presence of docosatetraenoic acid* in the molecule was detected after two weeks on this diet. Unlike the situation after feeding the FF diet, the FFL diet did not lead to an increase in 5,8,rr_eicosatrienoic acid in in the phosphatide. However, the FFL diet did not prevent the rapid loss of docosahexaenoic acid. The feeding of the hnolenic acid supplemented diet (FFLE) maintained a normal level of docosahexaenoic acid in this phosphatide and also led to the incorporation of appreciable amounts of eicosapentaenoic acid, which was found in trace levels in weanling and adult rat liver phosphatidylethanolamine. The FFLE diet was also effective in preventing a rise in 5,8,xreicosatrienoic acid, but was ineffective in preventing the large loss of arachidonic acid from the phosphatide over the 12 week period. Fatty acid comflosition of fihos@&idylinositol Very high levels of stearic acid (45-50x) and arachidonic acid (38-42%) were found in phosphatidylinositol that was isolated from weanling rat liver. Stearic acid content in this lipid molecule increased slightly on all three diets used in this study, but the most dramatic changes occurred in the levels of certain polyunsaturated fatty acids and these alterations are shown in Fig. 4. The FF diet resulted in a drastic reduction of arachidonic acid, along with a concomitant sharp increase in 5,8,11eicosatrienoic acid. Linoleic acid, which was present in phosphatidylinositol at the level of 6%, was reduced to trace levels on the fat-free diet. When linoleic acid was given along with the fat-free diet, there was very little alteration in the composition of this phosphatide. There was a small decrease in arachidonic acid during the initial weeks of this diet, but at the end of the 12 week period the level of this acid was approximately the same as that found in weanling animals. At the same time, there was a slight, but not pronounced increase in the content of 5,8,x1-eicosatrienoic acid. The daily supplementation of linolenic acid (FFLE) failed to prevent the loss of archidonic acid from the molecule, but the decline was more gradual than that observed on the FF diet. Docosahexaenoic acid, which was not detected in phosphatidylinositol isolated from either weanling or adult rat liver, did appear in this mole* Docosatetraenoic acid was tentatively identified by extrapolation of retention times of known standards but may be identical with the acid designated as 22 :5 o 6 by MORRHAUER AND HOLAWN‘. Biochim.
Biophys.
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g8 (1965) 502-5 I I
K. TISCHER, J. L. GLENN
‘0
I
FF
30 10 -----W_-.____.
FFL 40 YI \.-*A
*ITT•------.+----•
*o !O
,/‘.---+
..-.-
FFLE
‘0
WEEKS ON
DIETS
WEEKS
ON DIETS
Fig. 4. Major changes in fatty acid composition of phosphatidylinositol during FF, FFL and FFLE dietary periods. Symbols: O-0, arachidonic acid; O-O, eicosatrienoic acid; x-x, docosahexaenoic acid. Fig. 5. Major changes in fatty acid composition of phosphatidylserine during FF, FFL, and FFLE dietary periods. Symbols: o-0, arachidonic acid; x-x, docosahexaenoic acid; A-A, docosatetraenoic acid; O-C, eicosatrienoic acid; &----A, eicosapentaenoic acid.
cule as a result of the FFLE diet. Although not shown in Fig. 4, eicosa~n~~oic acid and docosapentaenoic acid were also found in phosphatidylinositol as a consequence of the daily administration of linolenic acid. Fatty acid com@xitiwt of phos$hatidylserine The fatty acid composition of this phosphatide, when it was isolated from weanling rat liver, was similar to that of phosphatidyleth~ol~e, differing mainly in the content of saturated rather than unsaturated fatty acids (Table I). The changes in the acyl components of this phosphatide as a result of the 3 diets (Fig. 5) were very much like those recorded for the ethanolamine containing lipid (Fig. 3). It should be stressed again, however, that both linoleic acid and linolenic acid supplementation to the fat free diet were equally effective in preventing an increase in 5,8,1x-eicosatrienoic acid in the molecule, even though the fatty acid patterns in this phosphatide were quite different as a result of the two diets. Fatty acid com$osition ofphos#hatidylcholitte The major changes which occurred in the first region of phosphatidylcholine are illustrated in Fig. 6. Although samples from the latter part of the phosphatidylcholine region were also analyzed, the results were similar to those presented in Fig. 6, and thus are not shown. On the FF diet, there was a large increase in the level of 5,8,rr-eicosatrienoic acid, while the levels of arachidonic acid and docosahexaenoic acid were sharply reBiochim.
Biophys.
Ada, 98 (rg65) 502-s I I
PHOSPHATIDE FATTY ACIDS OF ANIMALS ON FAT-FREE DIET
509
duced. An interesting finding was the decrease in pahnitic acid with an accompanying elevation of stearic acid (Table I). The overall decrease in the level of saturated acids was therefore minimized. Feeding the FFL diet prevented an increase in $8,rr-eicosatrienoic acid and also led to increases in arachidonic acid and docosatetraenoic acid. This diet was unable to prevent the rapid loss of docosahexaenoic acid from the phosphatide. The decrease in palmitic acid with a simultaneous increase in stearic acid, which was observed on the FF diet, also occurred during linoleic acid supplementation.
WEEKS
ON DIETS
Fig. 6. Major changes in fatty acid composition of phosphatidylcholine FFLE dietary periods. Symbols: O----O, arachidonic acid; X-X O-O, eicosatrienoic acid ; A-A, docosatetraenoic acid ; &---A,
I during FF. FFL, and , docosahexaenoic acid; eicosapentaenoic acid.
Linolenic acid supplementation (FFLE) led to changes in phosphatidylcholine which were quite similar to those already seen in the other phosphatides. The level of docosahexaenoic acid was maintained at a normal level, while at the same time, there was a sharp increase in eicosapentaenoic acid in the molecule. Arachidonic acid loss from this phosphatide (from 21% to 1.5%) wa.s even greater than that recorded for the animals on a fat free diet. A marked elevation of stearic acid, with a concomitant reduction of palmitic acid, was also observed to occur in phosphatidylcholine as a result of the FFLE diet. DISCUSSION
Numerous hypotheses have been proposed in an attempt to explain the biological function of linoleic acid, linolenic acid and their metabolic pro&r&, larly arachidonic acid and docosahexaenoic acid. Unfortunately, most of their postulated roles have encompassed complex biochemical processes rather than specific biochemical reactions. Thus, the maintenance of cell structurell, transport of serum lipids la, oxidative phosphorylationl*, electron transport1*, and other gross cellular Biochim.
Biophys.
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502-511
SO
K. TISCHER,
J. L. GLENN
phenomena have been sus+cted to depend to some degree on the presence of these acids, either singularly or when they are incorporated into molecules such as the phosphatides. The present study has made it possible to analyze the acyl groups of individual rat liver phosphatides during a period of rapid growth, when the animals were fed a fat-free diet or a fat-free diet with a daily supplementation of either linoleic acid or linolenic acid. As was expected, there were marked alterations in the fatty acid patterns of the phosphatides, but the observed changes were not the same for each phosphatide. For example, cardiolipin was rapidly depleted of its high linoleic acid content as a result of the fat-free diet, and this deficiency was counter balanced by large increases in oleic acid and palmitoleic acid. The replacement of linoleic acid by these monounsaturated acids, rather than by 5,8,rr-eicosatrienoic acid, was of interest since all of the other phosphatides exhibited large increases in 5,8,1x-eicosatrienoic acid when the animals were fed a fat-free diet. This acid has been shown to occur in high amounts in livers from rats on a fat-free diet, but the importance of analyzing individual phosphatides, rather than total lipid or total phospholipid, is thus stressed. The preferential incorporation of monoenoic acids into cardiolipin, rather than the polyenoic acid, could be an expression of the enzyme specificity involved in the biosynthetic acylation of cardiolipin. It would be of interest to determine if the cardiolipin molecule, as isolated from livers of animals fed a fat-free diet, still possessed the ability to activate the phosphatidase B enzyme of Penicillium notatumlb. The ability of either linoleic acid or linolenic acid supplementation to suppress the synthesis of 5,8,rr-eicosatrienoic acid was of interest, for it has been suggested that the synthesis of this acid is controlled by the amount of linoleic acid and arachidonic acid in the diet 16, or that both linoleic acid and linolenic acid are effective in inhibiting the synthesis of eicosatrienoic acid I’. In the present study the mechanism by which linolenic acid suppressed the formation of 5,8,rr_eicosatrienoic acid appeared to be different than that produced by linoleic acid supplementation. An analysis of the changes which occurred in phosphatidylethanolamine will serve to illustrate. Note that phosphatidylethanolamine, when obtained from weanling rat liver, contained high levels of arachidonic acid and docosahexaenoic acid. On the fat-free diet, the levels of these two acids decreased sharply and 5,8,rr-eicosatrienoic acid was then incorporated into the molecule in high amounts. The fatty acid patterns in this phosphatide were quite different as a consequence of supplementation with linoleic acid or linolenic acid, although both acids effectively blocked or markedly depressed the incorporation of 5,8,x1-eicosatrienoic acid. In the situation resulting from linoleic acid supplementation, an elevation in arachidonic acid content was observed and a previously lacking docosatetraenoic acid was detected. The latter acid is believed to be formed by a two-carbon addition to arachidonic acid and may be identical with “adrenic acid”, which was found in canine adrenal lipidsIn. It would appear that the synthesis of docosatetraenoic acid was an attempt by the animal to replace the docosahexaenoic acid content of phosphatidylethanolamine, which had decreased to a very low level. When linolenic acid was administered along with the fat-free diet, it was the arachidonic acid level in phosphatidylethanolamine which decreased precipitously while the level of docosahexaenoic acid increased slightly. Surprising, however, was the presence of high amounts of eicosapentaenoic acid in the molecule, which was normally present in only trace amounts in this phosphatide, when it was Biochim. Biophys. Acta, 98 (1965) 502-511
PHOSPHATIDE FATTY ACIDS OF ANIMALS ON FAT-FREE DIET
sr
isolated from weanling or adult rat liver. The results demonstrated that, in this situation, the animal preferred to replace archidonic acid with eicosapentaenoic rather than with 5,8,Ir_eicosatrienoic acid, and it could be that eicosapentaenoic acid is, like archidonic acid, capable of suppressing the synthesis of 5,8,rreicosatrienoic acid. The significance of the incorporation of an acid with five double bonds, in preference to an acid with three double bonds but of the same carbon length, is not understood at this time but could be dependent upon the double bond arrangement or degree of unsaturation. The present study has demonstrated the drastic changes which occurred in the fatty acyl composition of individual rat liver phosphatides as a result of a fat-free diet. The addition of either linoleic or linolenic acid to the fat-free diet provided the animal with a substrate which it could utilize to produce longer-chain polyunsaturated acids and to incorporate them into the phosphatides. The alterations in the phosphatides have undoubtedly changed their biochemical properties, but the mechanisms by which cellular metabolism is affected, are unknown at the present time. ACKNOWLEDGEMENTS The technical assistance of Mr. E. OPALKA and Mr. R. DASSO are gratefully acknowledged. This investigation was supported by U. S. Public Health Grants A 4711 and A 7714. The work of one of us (J. L. G.) was supported by a Research Career Development Award, GM-K8-14,018, Department of Health, Education and Welfare, Bethesda 14, Md.
REFERENCES I K. TISCHER AND J. L. GLENN, Federation Proc., 22 (x963) 377. 2 G. 0. BURR AND M. M. BURR, J. Biol. Ckm., 86 (1930) 587. 3 E. AAES-JBRGENSEN,Physiol. Revs., .+I (x96x) I. 4 J. F. MEAD, Federation PYOC.. 20 (x96x) 952. 5 H. MOHRHAUERAND R. T. HOLMAN. J. Lipid Res., 4 (1963) 151. 6 J. W. FARQUHAR,W. INSULL, Jr., P. ROSEN, W. STOPPELAND E. H. AHRENS, Jr., Nub. Rev., 17 (1959) Suppl. 8. 7 J. FOLCH, M. LEES AND G. H. SLOANE-STANLEY, J. Biol. Chcm., 226 (1957) 497. 8 J. L. GLENN, E. OPALKA AND K. TISCHBR, J. Biol. Chem., 238 (1963) 1249. g W. STO~EL. F. CHU AND E. H. AHRBNS, Jr., And. Chcm., 31 (1959) 307. IO L. C. A. NUNN AND I. SYEDLBY-MACLEAN, Biochem. J., 32 (1938) 2178. II G. 0. BURR, Federation PYOC.. I (1942) 224. 12 R. T. HOLMAN. Nuti. Abstv. Rev.. 16 (1~58) 3% . -_ , __ 13 P. D. KLEIN AND R. M. JOHNSON.J. Biol. Gem., 211 (1954) 103. 14 D. E. GREEN AND S. FLEISCAER,Biochem. Biophvs. Acta. 70 (1961) 554. - _. _ _ . 15 R. M. C. DAWSON, Biochem. J., 70 (Ig58c) 559: _ 16 R. PELUFFO, R. R. BRENNER AND 0. MERCURI, J. N&r., 81 (1963) IIO. 17 H. MOHRHAUERAND R. T. HOLMAN, J. N&r., 81 (1963) 67. 18 T. L. CHANG AND C. C. SWEELEY. Biochemistry, 2 (1963) 592. Biochim.
Biophys.
Abstr.
Acta, g8 (1965) 502-511
502 BBA 55107 THE FATTY ACIDS OF INDIVIDUAL GROWN
OX A FAT-FREE
LIVER PHOSPHATIDES
FROM RATS
DIET*
KONRAD TISCHER AND JOSEPH L. GLENN** Department (Received
of Biochemistry,
Albany
Medical College of Union University, Albany,
N. Y. (U.S.A.)
August 10th. 1964)
SUMMARY I. The purpose of the present study was to analyze the fatty acid composition of individual liver phosphatides from rats fed a fat-free diet for a r2-week period and commencing at the time of weaning. The effects of supplementation of either linoleic acid or linolenic acid to such a diet were also recorded. 2. The level of 5,8,rr-eicosatrienoic acid rose sharply in all phosphatides as a result of the fat-free diet, with the exception of cardiolipin, where large increases in oleic acid and palmitoleic acid were observed. 3. Daily supplementation of either linoleic acid or linolenic acid were effective in suppressing the synthesis of 5,8,x1-eicosatrienoic acid. The administration of linoleic acid with the fat-free diet resulted in the appearance of docosatetraenoic acid in some of the phosphatides, while linolenic acid supplementation led to the occurrence of eicosapentaenoic acid in all of the phosphatides.
INTRODUCTION It has been known for some time that rats, when reared on a fat-free diet, exhibited fatty acid patterns in their tissues which were different from the tissue fatty acids from rats that were grown on a normal stock diet. The earlier work of BURR AND BURR’ demonstrated that the symptoms of fat deficiency in the rat could be corrected by supplementation with fatty acids derived from lard. Recent reviews8Bphave discussed the dietary importance of both linoleic acid and linolenic acid, and a recent study by MOHBBAUERAND HOLMAN~illustrated how the dietary level of essential fatty acids affected the fatty acid composition of the total lipid fraction of rat liver. This study, although of considerable significance, did not permit the analyses of the fatty acids in the separate components of liver lipids. The primary intent of the present study was to analyze the fatty acid pattern of the individual phosphatides from rats, which were reared on a synthetic fat-free l Abbreviations: FF, rats fed with a fat free test diet; FFL, rats fed with a fat free test diet plus daily doses of linoleic acid methyl ester; FFLE, rats fed with a fat free test diet plus daily doses linolenic acid methyl ester. l l A preliminary report of this work has been published 1.
Biochim.
Biophys.
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diet from the time of weaning. In addition, the effects of daily supplementation of either linoleic acid or linolenic acid were also studied. The results of this study demonstrated the molecular changes that occurred in the liver phosphatides of rats on a fatfree diet and can be compared to the previously known changes that occurred on the tissue level. MATERIALS
AND METHODS
Animals All of the animals used in this study were female Wistar rats obtained from the Blue Spruce Animal Farms, Altamont, New York. The weights of these animals at the time of weaning were between 40-50 g. Diet and chemicals The basal diet was the Fat-Free Test Diet purchased from the Nutritional Biochemical Corporation, Cleveland, Ohio. Exhaustive extraction of samples of this diet with chloroform-methanol (z:I, v/v) demonstrated that the diet contained minute and insignificant traces of fatty acids. Linoleic acid methyl ester and linolenic acid methyl ester were obtained from Mann Chemical Research Company, New York, N.Y. Silicic acid and organic solvents of Analytical Reagent Grade were purchased from the Mallinckrodt Chemical Works, St. Louis, MO. The fatty acid methyl ester standards employed in gas-liquid chromatography were generously donated by the National Institutes of Health, Bethesda, hid., and Applied Science Labs, State College, Pa. All analyses of fatty acids were performed on a Barber-Coleman Model IO apparatus with a diethylene-glycol-succinate phase, coated on acid washed Chromosorb W (15.4% by weight, 80-100 mesh). The fatty acid composition of each phosphatide was determined by the triangulation method, and the values reported are as area per cent. Exfwimentd design A total of 107 female rats were used in this study. One group (FF) received only the fat-free test diet, while a second group (FFL) received the fat-free diet plus daily doses of linoleic acid methyl ester, and a third group (FFLE) received the fat-free diet plus daily supplementation of linolenic acid methyl ester. II weanling rats were used to obtain normal values and were designated as the control group. The livers from the control group were obtained immediately after decapitation and the total lipids were extracted and purified according to the method of FOLCH and co-workers?. Separation and identification of the individual phosphatides, and the methylation of the phosphatide fatty acids were performed as described previously’. Subsequently, the phosphatides were isolated from the livers of rats that were on their respective diets for I, 2, 3, 5.7, g and 12 weeks. The fatty acid pattern of each phosphatide was determined at these time intervals. At least four female rats were included at each time interval in each dietary group. 4 adult female rats, 14 weeks old, were raised on a normal stock diet and their liver phosphatide fatty acids were analyzed and served as comparative end values. The fat-free diet was available to the three dietary groups at all times. The daily Biochim.
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doses of linoieic acid and linolenic acid, as their methyl esters, were given orally with serological pipettes. The amount of each acid administered was 50 mg per day per rat for the iirst six weeks following weaning. Beginning with the seventh week and Iasting through the twelfth week, each rat received roe mg of the acid under study. Any of the rats which failed to eat the diet or showed signs of infection were discarded. 0.2 ml
RESULTS
The elution pattern of phosphatides off a silicic acid column is shown in Fig. I. The data illustrated were obtained from 5 g of liver which were removed from 3 weanling rats immediately after decapitation. 5 fractions were detected: viz. cardiolipin;
e TUBE NUMBER Fig. I. Fractionation of weanling rat liver phosphatides on siIicic acid column: CL, cardiolipin. PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; E’C I and PC II, initial and latter parts of phosphatidylcholine. The ratios 4/r and 312 refer to chloroform/ methanol ratios that were used to elute the phosphatides.
phosphatidylethanolamine; phosphatidylinositol; phosphatidyiserine; and phosphatidylcholine. The latter phosphatide was sub divided into 2 parts, because of previously demonstrated differences iu the fatty acid composition of the initial and latter parts of this fractions. A sixth rat liver phosphatide, sphingomyelin, could be eluted off the column with a higher concentration of methanol, but this compound was not analyzed in this investigation. The elution patterns of phosphatides from the rats on the 3 experimental diets showed little or no change from that of weanling rats, nor did the absolute amounts bmoles per g of wet liver) of the individual phosphatides vary significantly on any of the diets. There were, however, marked changes in the fatty acyl groups of the phosphatides during the 12 week periods, and these alterations were recorded by the following procedures. The 3 peak tubes of each fraction, with the exception of phosphatidylcholine, were combined and the phospholipid fatty acids that were present in these tubes were determined as their methyl esters* by gas-liquid chromatography. The phosphatidylcholine region was analyzed by detenniuiug the fatty acyl groups Biochim. Biophys.
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TABLE I FATTYACID COMPOSITION OF RAT LIVER PHOSPHATIDES AFTER 12 WEEK DIETARY PERIODS Com#ound Cardiolipin
Phosphatidylethanolamine
Diet*
Phosphatidylserine
Phosphatidylcholine I
Phosphatidylcholine II
16:r
18:o
18:1
18:a
4.7
2.0
78.5
30.1 11.2 14.8
1.7 2.8 2.3 5.3
13.2
6.6 5.6 9.9
44.3 29.0 40.8
7.2 38.2 3.0
22.0 12.5 16.4 17.2
trace 1.9 0.7 0.7
26.5 31.9 21.9 29.5
2.3 14.4 6.5 5.5
2.6 0.7 2.2 trace
W FF FFL FFLE
5.6 2.1
trace 5.7
45.4 53.3
2.6 6.5
5.8 0.9
3.9 3.3
0.5 2.0
45.0 46.7
2.8 6.1
W FF FFL FFLE
6.0 4.9
trace 5.1
40.2 44.7
4.1 3.5
1.3 1.5
W FF FFL FFLE
21.0 7.0 5.5 8.0
trace I.3 0.3 trace
W FF FFL FFLE W FF FFL
FFLE Phosphatidylinositol
16:o
W FF
FFL FFLE
x8:3
aor
ao:q
20:s
22:4
22:5
2a:6
-
trace -
1.1 13.5
4.0 5.9 0.7
trace 3.8 4.0 trace
trace 1.5
-
-
trace
21.0 1.6
20.8 10.7 38.7 3.2
trace 15.0
trace 10.8 -
2.0 0.7
-
1.1 29.4
38.4 2.3
-
-
-
-
1.8
39.1 6.3
5.1 10.4
5.1 trace
-
14.9
24.9 10.3
-
46.8 46.7
5.1 6.8
1.5 0.3
trace 2.6
31.1 3.1
-
8.0
-
29.3 34.3 35.6
3.8 11.6 5.1 6.6
3.8 trace I.9 -
-
34.0 1.3 3.6
21.1 6.0 35.6
trace 16.8
0.5 0.4
1.5
7.5
-
1.0
9.0 18.1
0.9
6.5 I.2 26.7
-
1.0
20.2
1.9 10.2 -
trace
4.3 3.5 21.0
-
-
0.8
15.4
8.9
15.0
-
-
15.7
-
-
6.1 2.4 3.9
23.9 21.8 24.3
26.9 13.7 23.2
trace 8.2 0.6
-
I7.7 0.8 2.0
3.9 26.0 1.6
13.5
-
8.5
2.3 -
-
* W, phosphatides were isolated from weanling rats.
in 3 tubes in the middle of the ascending slope (PC I), and in 3 tubes in the middle of the descending slope (PC II). It was decided that the sampling techniques described above, rather than the combining of all the tubes in each fraction, would yield more accurate data and also avoid any overlap between individual components. The fatty acid changes which occurred in the individual phosphatides are discussed separately in the following sections, but an overall comparison in the fatty acyl composition of phospholipids from weanling rat liver and in the livers from rats which were on their respective diets for 12 weeks is presented in Table I. Fatty acid com$osition of cardiolifiin This molecule was characterized in weanling rat liver, as well as in adult rat liver, by having a very high level of linoleic acid (78%) and appreciable amounts of oleic acid (13%). Major changes which occurred in the fatty acyl content of cardiolipin during the 12 week dietary periods, are shown in Fig. 2. The most drastic alteration in this component, when the rats were placed on the FF diet, was the precipitous fall in the level of linoleic acid which decreased to 22% after only I week on the diet. After 12 weeks, the linoleic acid content of cardiolipin was 7%. To compensate for this loss, there were large increases in the levels of oleic acid (from 13% to 44%), and of pahnitoleic acid (from 2% to 30%) at the end of 12 Biofihys.
23.3 7.0 3.5 26. I
trace 1.2
33.5
Biochim.
10.7
2.9
-
21.6 25.4 23.3
0.4 1.5
-
-
-
4.3 16.5
-
-
42.3
2.5
trace
Acta, 98 (1965) 502-511
9.6 0.4 6.8
K. TISCHER, J. L. GLENN
‘0 la
FF
I
FF (0
WEEKS ON WETS Fig. 2. Major changes in fatty acid composition periods. Symbols: o-o, linoleic acid; x-x docosahexaenoic acid.
WEEKS
ON DIETS
of cardiolipin during FF, FFL, and FFLE dietary , oleic acid; O-O, palmitoleic acid: A-A,
Fig. 3. Major changes in fatty acid composition of phosphatidylethanolamine during FF. FFL, and FFLE dietary periods, Symbols: o-0, arachidonic acid; x -x , docosahexaenoic acid; O-C, docosatetraenoic acid; A-A, eicosatrienoic acid; A---A, eicosapentaenoic acid.
weeks. There was only a small amount of eicosatrienoic acid* incorporated into cardiolipin as a consequence of the FF diet, although the livers of animals on such a diet are known to have high concentrations of this acidlo. When the rats were raised on the FFL diet, there was still a decrease in the level of linoleic acid in cardiolipin, but it was not nearly as pronounced as that seen on the FF diet. Apparently, the level of linoleic acid that was administered, 50 mg in ester form per day for the first 6 weeks, and IOO mg per day for the remaining weeks, was not sufficient to maintain a normal level of this acid in cardiolipin. The decrease observed was accompanied by elevation of oleic acid and palmitoleic acid. The third diet (FFLE), that of linolenic acid methyl ester supplementation to an otherwise fat-free diet, resulted in an almost complete disappearance of linoleic acid from cardiolipin. Concomitant with this loss, there were large increases in oleic acid and palmitoleic acid, as well as the appearance of linolenic acid, docosapentaenoic acid and docosahexaenoic acid in the molecule. The last three acids were present in trace amounts in cardiolipin from weanling and adult rat liver. Fatty acid composition of phosphatidyletha+whzmine In weanling rat liver, this phospholipid contained high levels of palmitic acid (22%), stearic acid (26%), arachidonic acid (21%) and docosahexaenoic acid (23%).
l 5,8,1x-eicosatrienoic acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexanoic acid were identified by relative retention times and‘the use of standards.
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All of these acids, with the exception of stearic acid, decreased in phosphatidylethanolamine when the animals were placed on the FF diet. However, the most interesting changes were observed in the levels of the polyunsaturated acids and they are recorded in Fig. 3. along with the alterations found in this phosphatide as a consequence of the other diets. On the FF diet, arachidonic decreased gradually while the level of docosahexaenoic acid dropped sharply and appeared to stabilize at approx. 5%. The above losses were compensated for by a marked elevation in 5,8,rr-eicosatrienoic acid, which was not present in weanling rat liver phosphatidylethanolamine, to a level of 21% after 12 weeks. Although not shown in Fig. 3, oleic acid was also elevated from 2% to 14% at the conclusion of the FF dietary period. In the FFL animals, the level of linoleic acid in phosphatidylethanolamine did not change significantly, but arachidonic acid content increased steadily to reach a level of 39% after the 12 week period. The presence of docosatetraenoic acid* in the molecule was detected after two weeks on this diet. Unlike the situation after feeding the FF diet, the FFL diet did not lead to an increase in 5,8,rr_eicosatrienoic acid in in the phosphatide. However, the FFL diet did not prevent the rapid loss of docosahexaenoic acid. The feeding of the hnolenic acid supplemented diet (FFLE) maintained a normal level of docosahexaenoic acid in this phosphatide and also led to the incorporation of appreciable amounts of eicosapentaenoic acid, which was found in trace levels in weanling and adult rat liver phosphatidylethanolamine. The FFLE diet was also effective in preventing a rise in 5,8,xreicosatrienoic acid, but was ineffective in preventing the large loss of arachidonic acid from the phosphatide over the 12 week period. Fatty acid comflosition of fihos@&idylinositol Very high levels of stearic acid (45-50x) and arachidonic acid (38-42%) were found in phosphatidylinositol that was isolated from weanling rat liver. Stearic acid content in this lipid molecule increased slightly on all three diets used in this study, but the most dramatic changes occurred in the levels of certain polyunsaturated fatty acids and these alterations are shown in Fig. 4. The FF diet resulted in a drastic reduction of arachidonic acid, along with a concomitant sharp increase in 5,8,11eicosatrienoic acid. Linoleic acid, which was present in phosphatidylinositol at the level of 6%, was reduced to trace levels on the fat-free diet. When linoleic acid was given along with the fat-free diet, there was very little alteration in the composition of this phosphatide. There was a small decrease in arachidonic acid during the initial weeks of this diet, but at the end of the 12 week period the level of this acid was approximately the same as that found in weanling animals. At the same time, there was a slight, but not pronounced increase in the content of 5,8,x1-eicosatrienoic acid. The daily supplementation of linolenic acid (FFLE) failed to prevent the loss of archidonic acid from the molecule, but the decline was more gradual than that observed on the FF diet. Docosahexaenoic acid, which was not detected in phosphatidylinositol isolated from either weanling or adult rat liver, did appear in this mole* Docosatetraenoic acid was tentatively identified by extrapolation of retention times of known standards but may be identical with the acid designated as 22 :5 o 6 by MORRHAUER AND HOLAWN‘. Biochim.
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K. TISCHER, J. L. GLENN
‘0
I
FF
30 10 -----W_-.____.
FFL 40 YI \.-*A
*ITT•------.+----•
*o !O
,/‘.---+
..-.-
FFLE
‘0
WEEKS ON
DIETS
WEEKS
ON DIETS
Fig. 4. Major changes in fatty acid composition of phosphatidylinositol during FF, FFL and FFLE dietary periods. Symbols: O-0, arachidonic acid; O-O, eicosatrienoic acid; x-x, docosahexaenoic acid. Fig. 5. Major changes in fatty acid composition of phosphatidylserine during FF, FFL, and FFLE dietary periods. Symbols: o-0, arachidonic acid; x-x, docosahexaenoic acid; A-A, docosatetraenoic acid; O-C, eicosatrienoic acid; &----A, eicosapentaenoic acid.
cule as a result of the FFLE diet. Although not shown in Fig. 4, eicosa~n~~oic acid and docosapentaenoic acid were also found in phosphatidylinositol as a consequence of the daily administration of linolenic acid. Fatty acid com@xitiwt of phos$hatidylserine The fatty acid composition of this phosphatide, when it was isolated from weanling rat liver, was similar to that of phosphatidyleth~ol~e, differing mainly in the content of saturated rather than unsaturated fatty acids (Table I). The changes in the acyl components of this phosphatide as a result of the 3 diets (Fig. 5) were very much like those recorded for the ethanolamine containing lipid (Fig. 3). It should be stressed again, however, that both linoleic acid and linolenic acid supplementation to the fat free diet were equally effective in preventing an increase in 5,8,1x-eicosatrienoic acid in the molecule, even though the fatty acid patterns in this phosphatide were quite different as a result of the two diets. Fatty acid com$osition ofphos#hatidylcholitte The major changes which occurred in the first region of phosphatidylcholine are illustrated in Fig. 6. Although samples from the latter part of the phosphatidylcholine region were also analyzed, the results were similar to those presented in Fig. 6, and thus are not shown. On the FF diet, there was a large increase in the level of 5,8,rr-eicosatrienoic acid, while the levels of arachidonic acid and docosahexaenoic acid were sharply reBiochim.
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duced. An interesting finding was the decrease in pahnitic acid with an accompanying elevation of stearic acid (Table I). The overall decrease in the level of saturated acids was therefore minimized. Feeding the FFL diet prevented an increase in $8,rr-eicosatrienoic acid and also led to increases in arachidonic acid and docosatetraenoic acid. This diet was unable to prevent the rapid loss of docosahexaenoic acid from the phosphatide. The decrease in palmitic acid with a simultaneous increase in stearic acid, which was observed on the FF diet, also occurred during linoleic acid supplementation.
WEEKS
ON DIETS
Fig. 6. Major changes in fatty acid composition of phosphatidylcholine FFLE dietary periods. Symbols: O----O, arachidonic acid; X-X O-O, eicosatrienoic acid ; A-A, docosatetraenoic acid ; &---A,
I during FF. FFL, and , docosahexaenoic acid; eicosapentaenoic acid.
Linolenic acid supplementation (FFLE) led to changes in phosphatidylcholine which were quite similar to those already seen in the other phosphatides. The level of docosahexaenoic acid was maintained at a normal level, while at the same time, there was a sharp increase in eicosapentaenoic acid in the molecule. Arachidonic acid loss from this phosphatide (from 21% to 1.5%) wa.s even greater than that recorded for the animals on a fat free diet. A marked elevation of stearic acid, with a concomitant reduction of palmitic acid, was also observed to occur in phosphatidylcholine as a result of the FFLE diet. DISCUSSION
Numerous hypotheses have been proposed in an attempt to explain the biological function of linoleic acid, linolenic acid and their metabolic pro&r&, larly arachidonic acid and docosahexaenoic acid. Unfortunately, most of their postulated roles have encompassed complex biochemical processes rather than specific biochemical reactions. Thus, the maintenance of cell structurell, transport of serum lipids la, oxidative phosphorylationl*, electron transport1*, and other gross cellular Biochim.
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SO
K. TISCHER,
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phenomena have been sus+cted to depend to some degree on the presence of these acids, either singularly or when they are incorporated into molecules such as the phosphatides. The present study has made it possible to analyze the acyl groups of individual rat liver phosphatides during a period of rapid growth, when the animals were fed a fat-free diet or a fat-free diet with a daily supplementation of either linoleic acid or linolenic acid. As was expected, there were marked alterations in the fatty acid patterns of the phosphatides, but the observed changes were not the same for each phosphatide. For example, cardiolipin was rapidly depleted of its high linoleic acid content as a result of the fat-free diet, and this deficiency was counter balanced by large increases in oleic acid and palmitoleic acid. The replacement of linoleic acid by these monounsaturated acids, rather than by 5,8,rr-eicosatrienoic acid, was of interest since all of the other phosphatides exhibited large increases in 5,8,1x-eicosatrienoic acid when the animals were fed a fat-free diet. This acid has been shown to occur in high amounts in livers from rats on a fat-free diet, but the importance of analyzing individual phosphatides, rather than total lipid or total phospholipid, is thus stressed. The preferential incorporation of monoenoic acids into cardiolipin, rather than the polyenoic acid, could be an expression of the enzyme specificity involved in the biosynthetic acylation of cardiolipin. It would be of interest to determine if the cardiolipin molecule, as isolated from livers of animals fed a fat-free diet, still possessed the ability to activate the phosphatidase B enzyme of Penicillium notatumlb. The ability of either linoleic acid or linolenic acid supplementation to suppress the synthesis of 5,8,rr-eicosatrienoic acid was of interest, for it has been suggested that the synthesis of this acid is controlled by the amount of linoleic acid and arachidonic acid in the diet 16, or that both linoleic acid and linolenic acid are effective in inhibiting the synthesis of eicosatrienoic acid I’. In the present study the mechanism by which linolenic acid suppressed the formation of 5,8,rr_eicosatrienoic acid appeared to be different than that produced by linoleic acid supplementation. An analysis of the changes which occurred in phosphatidylethanolamine will serve to illustrate. Note that phosphatidylethanolamine, when obtained from weanling rat liver, contained high levels of arachidonic acid and docosahexaenoic acid. On the fat-free diet, the levels of these two acids decreased sharply and 5,8,rr-eicosatrienoic acid was then incorporated into the molecule in high amounts. The fatty acid patterns in this phosphatide were quite different as a consequence of supplementation with linoleic acid or linolenic acid, although both acids effectively blocked or markedly depressed the incorporation of 5,8,x1-eicosatrienoic acid. In the situation resulting from linoleic acid supplementation, an elevation in arachidonic acid content was observed and a previously lacking docosatetraenoic acid was detected. The latter acid is believed to be formed by a two-carbon addition to arachidonic acid and may be identical with “adrenic acid”, which was found in canine adrenal lipidsIn. It would appear that the synthesis of docosatetraenoic acid was an attempt by the animal to replace the docosahexaenoic acid content of phosphatidylethanolamine, which had decreased to a very low level. When linolenic acid was administered along with the fat-free diet, it was the arachidonic acid level in phosphatidylethanolamine which decreased precipitously while the level of docosahexaenoic acid increased slightly. Surprising, however, was the presence of high amounts of eicosapentaenoic acid in the molecule, which was normally present in only trace amounts in this phosphatide, when it was Biochim. Biophys. Acta, 98 (1965) 502-511
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isolated from weanling or adult rat liver. The results demonstrated that, in this situation, the animal preferred to replace archidonic acid with eicosapentaenoic rather than with 5,8,Ir_eicosatrienoic acid, and it could be that eicosapentaenoic acid is, like archidonic acid, capable of suppressing the synthesis of 5,8,rreicosatrienoic acid. The significance of the incorporation of an acid with five double bonds, in preference to an acid with three double bonds but of the same carbon length, is not understood at this time but could be dependent upon the double bond arrangement or degree of unsaturation. The present study has demonstrated the drastic changes which occurred in the fatty acyl composition of individual rat liver phosphatides as a result of a fat-free diet. The addition of either linoleic or linolenic acid to the fat-free diet provided the animal with a substrate which it could utilize to produce longer-chain polyunsaturated acids and to incorporate them into the phosphatides. The alterations in the phosphatides have undoubtedly changed their biochemical properties, but the mechanisms by which cellular metabolism is affected, are unknown at the present time. ACKNOWLEDGEMENTS The technical assistance of Mr. E. OPALKA and Mr. R. DASSO are gratefully acknowledged. This investigation was supported by U. S. Public Health Grants A 4711 and A 7714. The work of one of us (J. L. G.) was supported by a Research Career Development Award, GM-K8-14,018, Department of Health, Education and Welfare, Bethesda 14, Md.
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