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Dietary linoleic acid and the accumulation of homo-γ-linolenic acid in the cholesterol esters of the adrenal gland of the rabbit
PRELIMINARY
18r
NOTES
Dietary linoleic acid and the accumulation of homo-y-linofenic && cholesterol esters of the adrenal gland of the rabbit
acid in
In most tissues of the rabbit that have been studied so far the concentrations of C,, and C,, polyenoic acids are much lower than in the same tissues of other species1-3. The concentrations of these acids in the liver and plasma of the rabbit cannot be increased by raising the level of linoleic acid in the diet %a. DAILEY et d4 have shown, however, that the cholesterol esters of the adrenal glands do contain much higher levels of these long-chain poly-unsaturated acids than are found in other tissues of the rabbit. In this communication the preliminary results of an investigation into the effects of changes in diet on the cholesterol ester fatty acids of the rabbit adrenal gland are presented. The report is concerned primarily with the identification of an eicosatrienoic acid that accumulates in the adrenal cholesterol esters of rabbits given diets containing high levels of linoleic acid. 5 groups of male, New Zealand white rabbits, 6 months of age, were given for 38 weeks a diet consisting of 80 parts of a semi-synthetic basal diet to which were added: for Group I, 20 parts maize-oil; for Group 2, 20 parts butterfat; for Group 3, TO parts maize-oil and IO parts butterfat; for Group 4, 0.47 parts maize-oil and 43 parts wheat starch; and for Group 5, 10.1 parts maize-oil and 21.6 parts wheat starch. A 6th group was maintained on a commercial rabbit diet. Fuller details of rabbits and diets have been published elsewhere 5. The linoleic acid contents of the diets are given in Table I. After the rabbits had been killed the adrenal glands of 6 rabbits in each group were removed. The glands were pooled in such a way that for each dietary treatment there were 3 bulked groups, each bulked group containing the adrenal glands from 2 rabbits. The total lipids from each pooled group were extracted in chloroform-methanol (Z : I, v/v) and the cholesterol ester fraction was isolated by chromatography on columns of silicic acid. Methyl esters of the cholesterol ester fatty acids were prepared by transesterification and subjected to gas-liquid TABLE THE
I
COWPOSITION
CHOLESTEROL
(PERCENTAGE
ESTERS
FROM
THE
OF
THE
TOTAL)
EXPERIMENTAL
OF
THE
MAJOR
FATTY
ACIDS
OF
THE
ADRENAL
RABBITS
Tbe linoleic acid contents of the diets (g/100 g) in the different groups are as EoIlows: I, 10.6; z, 0.27; 3, 5.4; 4, 0.21; 5, 4.9; 6, 1.9. The percentage composition is given in mean values with their standard errors. Fatty
acids
--Saturated acids lMonoenoic acids r8:2
zo:3 (I) zo:3 (2) 20:4 22:4 Abbreviations:
Group
Gro@
I
(%)
2
(73
21.1 f
1.83
23.8 & 0.64
44.0 9.4 I.1 9.0 6.9 4.9
1.70 0.90 0.07 0.90 0.99 0.59
59.6 2.0 0.9 2.3
It k * & * j,
PEGA,
i: & & &
20.0 f
0.45
1.05 46.9 rrt 2.31 0.09 9.0 + 0.94 O.LO 0.6 si_:0.09 0.20 6.0 i: 0.79 1.8 f 0.29 5.5 i_ I.11 3.0 + 0.25 5.1 * 1.23
IO% poIyethylene
Group 4 (%)
Grmp
(%)
(%I
19.2 It 3.57
15.6 Ifr:0.81
22.0 & 1.03
55.8 3.6 1.5 2.6 4-3 5.6
1.46 0.26 0.06
46.5 xt 1.57 7.0 & 1.62 1.0 & 0.1%
0.52
5.2 rrt 0.93 10.0 ;t 0.38 6.9 & 0.60
44.5 6.7 0.6 4.x 5-4 5.6
glycol adipate;
rt + & & + +
0.55 1.31
APL,
Group 6
5
i + & + zk ”
2.85 0.06 0.16 0.35 0.73 1.03
10% Apiezon L.
Bioclzim. Bio$Gqw. A&,
x16 (1966) r&-r83
chromatograpl~~7 on both the polar PEGA and the non-polar APL columns. Detaiis of the methods used in these procedures have been published eisewherez,R~S. Preliminary identification of the fatty acids was carried out by comparison of retention times with those of known standard acids, and by plotting the vaiues of the logarithms of the retention times on the polar PEGA column against those on the non-polar APL coiumn6. Total methyl ester samples were chromatographed on thin-layer plates of silica gel G-silver nitrate (95: 5, or 90: IO, -w/w)’using a mixture of light petroleum (b.p. 40-60”) and diethyl ether (85 : 15, v/v) as the developing solvent. The methyl esters were separated by this technique into saturated, monoenoic, dienoic, trienoic, and other polyenoic acid esters and the respective zones were scraped from the plate. The fractions were then eluted from the various bands of silica gel G with h~xane-d~ethyl ether (50 : 50, v/v) and analysed by gas-chromatography as an identification check. Certain of the methyl ester fractions were subjected to oxidation (CHANG AND SWEEUZY~) and the resultant dicarboxyk acids analysed by gas-chromatography on coltimns of APL or PEGa at a temperature of 175O. The results of the fatty acid analyses (Table I) show that the adrenal cholesterol esters of each group of rabbits contained appreciable amounts (3.0-6.9%) of a docosatetraenoic acid, presumably adrenlc acid. DAILEY et al.” found oniy trace amounts of this acid in the adrenai cholesterol esters of the rabbit, in contrast to other species studied by them. The other noticeable feature of Table 1 is the presence of z isomers of eicosatrienoic acid, zo : 3 (I) and 20 : 3 j2). The concentration of the major isomer, zo:3 (z), appeared to be correlated with the level of dietaq i hnoleic acid, increasing progressively from 2,374 in the adrenal cholesterol esters of the rabbits given a diet containing o.z~*/~ linoleic acid (Group 2) to 9% in those of the rabbits given a diet containing lo.O"/,linoleic acid (Group I). The other isomer, 20: 3 (I), which had a lower retention time than 20 : 3 (2) on bcth APL a,nd PEGA columns, occurred in IOW concentrations in all 6 groups of rabbits. TABLE THE
XI
COMPOSITION
FROM
THEIR
Trim&
OF
THE
OXIDATION,
acids
{ “/. of tafnl
TRIESOIC OF THE
( “4
{
oJr
TOGETHER CHOLESTEROL
Ricadxwyylic
tvimesj
c5
;:i*
C, Ci
CS C, C I” * Possible contamination
WITH
THE
ESTERS
DICARBOXYLIC
ACIDS
FROM THE RABBITS ..__ -__.
OBTAINED
OF GROVE’
I
acids
~~~~~) ~-
--_
Gs
ACIDS,
ADREPiL
-.--_-
____I
__---._
3-9
I.0
9.3 85.9 not detected 0.0
with octadecadienoic
acid.
Table II shows the fatty acid composition of the trienoic acid fraction obtained from a pooled sample of adrenal cholesterol esters from the rabbits of Group I. The table also gives the composition of the dicarboxyhc acids obtained by oxidation of this fraction. The major trienoic acid present gave rise to the C, dicarboxylk acid, acid, homo-~-~~ole~~ acid, which suberic acid, and is therefore d 8~11~~4-eicosat~~eno~c Biockiun.Biophys.
Acta,
I 16 (1966) 181-183
PRELIMINARY
NOTES
183
is derived from linoleic acid g. It is not possible from these results alone to deduce with certainty the structure of the other isomer, 20: 3 (I), but comparison of the “equivalent chain lengths” of the z isomers with the tabulated values given by HOFSTETTER et al.10 indicate that this acid is A VTll-eicosatrienoic acid which is known to be derived from oleic acids. GIDEZ~’ has recently demonstrated the presence of 2 isomeric eicosatrienoic acids in the adrenal cholesterol esters of the rat. Tentative identification, based on gas-chromatographic retention properties only, suggested that I isomer, present in small amounts in normal rats, was probably As~l~14-eicosatrienoic acid, whereas the other isomer, present only in rats deficient in essential fatty acid was probably A578711-eicosatrienoicacid. RILEY I2 has also suggested on theoretical grounds that an eicosatrienoic acid present in human adrenal glands might be the 118,11,14isomer. As far as we are aware the results presented in this communication are the acid in more than first to show conclusively the presence of A 8>11>14-eicosatrienoic trace quantities in rabbit tissues. High levels of this acid have however been reported in the cholesterol esters of canine adrenals 8p13.The accumulation of this acid in the adrenal cholesterol esters of rabbits given diets containing high levels of linoleic acid is in marked contrast to the many reports of an accumulation of eicosatrienoic acid in various tissues of animals given essential fatty acid-deficient diets. In view of these findings a note of caution should perhaps be sounded against the unqualified use of the trienoic acid: tetraenoic acid ratio as a diagnostic test for essential fatty acid status 14,15,particularly in studies involving the rabbit. J. H. MOORE D. L. WILLIAMS
National Institute fey Research in Dairy&g, ShinJield, Reading (Great Britain) I L. SWELL AND C. origin, Academic, 2 J. H. MOORE AND 3 J. H. MOORE AND 4 R. E. DAILEY, L.
R. TREADWELL, in M. SANDLER AND G. H. BOURNE, Athwosclerosis and its New York, 1963, p. 301. D. L. WILLIAMS, Ca?z. J. Biochenz. Physiol., qr (1963) 1821. D. L. WILLIAMS, B&t. J. N&r., 18 (1964) 603. SWELL, H. FIELD AND C. R. TREADWELL, PYOC. Sm. Exptl. Biol. Med., 105
(1960) 4. 5 J. H. MOORE AND D. L. WILLIAMS, Brit. J. N&r., 18 (1964) 253. 6 A. T. JAMES, J. Chvonzatog., z (1959) 552. 7 L. J. MORRIS, Chem. Ind. (Lo?zdon), (1962) 1238. 8 T. L. CHANG AND C. C. SWEELEY, r’. L&d Res., 3 (1962) 170. 9 J. F. MEAD, in K. BLOCH, Lipid A?etab&sm, Wile<, New York, 1960, p. 41. IO H. H. HOFSTETTER, N. SEN AND R. T. HOLMAN, I. Am. Oil Chavnists’ Sac.. 42 (1965) 577. , _ -, --, I I L. I. GIDEZ, Biochem. Biophys. Res. Commun., 14~(1964) 413. 12 C. RILEY, Biochem. J., 87 (7963) 500. 13 T. L. CHANG AND C. C. SWEELEY, Biochemistry, 2 (1963) 592. 14 H. F. WIESE, A. E. HANSEN AND D. J. D. ADAM, J. Nutv., 66 (1958) 345. 15 R. T. HOLMAN, J. N&r., 70 (1960) 405.
Received August 4th, 1965. Biochivn.
Biophys.
Acta,
116 (1966) 181-183
18r
NOTES
Dietary linoleic acid and the accumulation of homo-y-linofenic && cholesterol esters of the adrenal gland of the rabbit
acid in
In most tissues of the rabbit that have been studied so far the concentrations of C,, and C,, polyenoic acids are much lower than in the same tissues of other species1-3. The concentrations of these acids in the liver and plasma of the rabbit cannot be increased by raising the level of linoleic acid in the diet %a. DAILEY et d4 have shown, however, that the cholesterol esters of the adrenal glands do contain much higher levels of these long-chain poly-unsaturated acids than are found in other tissues of the rabbit. In this communication the preliminary results of an investigation into the effects of changes in diet on the cholesterol ester fatty acids of the rabbit adrenal gland are presented. The report is concerned primarily with the identification of an eicosatrienoic acid that accumulates in the adrenal cholesterol esters of rabbits given diets containing high levels of linoleic acid. 5 groups of male, New Zealand white rabbits, 6 months of age, were given for 38 weeks a diet consisting of 80 parts of a semi-synthetic basal diet to which were added: for Group I, 20 parts maize-oil; for Group 2, 20 parts butterfat; for Group 3, TO parts maize-oil and IO parts butterfat; for Group 4, 0.47 parts maize-oil and 43 parts wheat starch; and for Group 5, 10.1 parts maize-oil and 21.6 parts wheat starch. A 6th group was maintained on a commercial rabbit diet. Fuller details of rabbits and diets have been published elsewhere 5. The linoleic acid contents of the diets are given in Table I. After the rabbits had been killed the adrenal glands of 6 rabbits in each group were removed. The glands were pooled in such a way that for each dietary treatment there were 3 bulked groups, each bulked group containing the adrenal glands from 2 rabbits. The total lipids from each pooled group were extracted in chloroform-methanol (Z : I, v/v) and the cholesterol ester fraction was isolated by chromatography on columns of silicic acid. Methyl esters of the cholesterol ester fatty acids were prepared by transesterification and subjected to gas-liquid TABLE THE
I
COWPOSITION
CHOLESTEROL
(PERCENTAGE
ESTERS
FROM
THE
OF
THE
TOTAL)
EXPERIMENTAL
OF
THE
MAJOR
FATTY
ACIDS
OF
THE
ADRENAL
RABBITS
Tbe linoleic acid contents of the diets (g/100 g) in the different groups are as EoIlows: I, 10.6; z, 0.27; 3, 5.4; 4, 0.21; 5, 4.9; 6, 1.9. The percentage composition is given in mean values with their standard errors. Fatty
acids
--Saturated acids lMonoenoic acids r8:2
zo:3 (I) zo:3 (2) 20:4 22:4 Abbreviations:
Group
Gro@
I
(%)
2
(73
21.1 f
1.83
23.8 & 0.64
44.0 9.4 I.1 9.0 6.9 4.9
1.70 0.90 0.07 0.90 0.99 0.59
59.6 2.0 0.9 2.3
It k * & * j,
PEGA,
i: & & &
20.0 f
0.45
1.05 46.9 rrt 2.31 0.09 9.0 + 0.94 O.LO 0.6 si_:0.09 0.20 6.0 i: 0.79 1.8 f 0.29 5.5 i_ I.11 3.0 + 0.25 5.1 * 1.23
IO% poIyethylene
Group 4 (%)
Grmp
(%)
(%I
19.2 It 3.57
15.6 Ifr:0.81
22.0 & 1.03
55.8 3.6 1.5 2.6 4-3 5.6
1.46 0.26 0.06
46.5 xt 1.57 7.0 & 1.62 1.0 & 0.1%
0.52
5.2 rrt 0.93 10.0 ;t 0.38 6.9 & 0.60
44.5 6.7 0.6 4.x 5-4 5.6
glycol adipate;
rt + & & + +
0.55 1.31
APL,
Group 6
5
i + & + zk ”
2.85 0.06 0.16 0.35 0.73 1.03
10% Apiezon L.
Bioclzim. Bio$Gqw. A&,
x16 (1966) r&-r83
chromatograpl~~7 on both the polar PEGA and the non-polar APL columns. Detaiis of the methods used in these procedures have been published eisewherez,R~S. Preliminary identification of the fatty acids was carried out by comparison of retention times with those of known standard acids, and by plotting the vaiues of the logarithms of the retention times on the polar PEGA column against those on the non-polar APL coiumn6. Total methyl ester samples were chromatographed on thin-layer plates of silica gel G-silver nitrate (95: 5, or 90: IO, -w/w)’using a mixture of light petroleum (b.p. 40-60”) and diethyl ether (85 : 15, v/v) as the developing solvent. The methyl esters were separated by this technique into saturated, monoenoic, dienoic, trienoic, and other polyenoic acid esters and the respective zones were scraped from the plate. The fractions were then eluted from the various bands of silica gel G with h~xane-d~ethyl ether (50 : 50, v/v) and analysed by gas-chromatography as an identification check. Certain of the methyl ester fractions were subjected to oxidation (CHANG AND SWEEUZY~) and the resultant dicarboxyk acids analysed by gas-chromatography on coltimns of APL or PEGa at a temperature of 175O. The results of the fatty acid analyses (Table I) show that the adrenal cholesterol esters of each group of rabbits contained appreciable amounts (3.0-6.9%) of a docosatetraenoic acid, presumably adrenlc acid. DAILEY et al.” found oniy trace amounts of this acid in the adrenai cholesterol esters of the rabbit, in contrast to other species studied by them. The other noticeable feature of Table 1 is the presence of z isomers of eicosatrienoic acid, zo : 3 (I) and 20 : 3 j2). The concentration of the major isomer, zo:3 (z), appeared to be correlated with the level of dietaq i hnoleic acid, increasing progressively from 2,374 in the adrenal cholesterol esters of the rabbits given a diet containing o.z~*/~ linoleic acid (Group 2) to 9% in those of the rabbits given a diet containing lo.O"/,linoleic acid (Group I). The other isomer, 20: 3 (I), which had a lower retention time than 20 : 3 (2) on bcth APL a,nd PEGA columns, occurred in IOW concentrations in all 6 groups of rabbits. TABLE THE
XI
COMPOSITION
FROM
THEIR
Trim&
OF
THE
OXIDATION,
acids
{ “/. of tafnl
TRIESOIC OF THE
( “4
{
oJr
TOGETHER CHOLESTEROL
Ricadxwyylic
tvimesj
c5
;:i*
C, Ci
CS C, C I” * Possible contamination
WITH
THE
ESTERS
DICARBOXYLIC
ACIDS
FROM THE RABBITS ..__ -__.
OBTAINED
OF GROVE’
I
acids
~~~~~) ~-
--_
Gs
ACIDS,
ADREPiL
-.--_-
____I
__---._
3-9
I.0
9.3 85.9 not detected 0.0
with octadecadienoic
acid.
Table II shows the fatty acid composition of the trienoic acid fraction obtained from a pooled sample of adrenal cholesterol esters from the rabbits of Group I. The table also gives the composition of the dicarboxyhc acids obtained by oxidation of this fraction. The major trienoic acid present gave rise to the C, dicarboxylk acid, acid, homo-~-~~ole~~ acid, which suberic acid, and is therefore d 8~11~~4-eicosat~~eno~c Biockiun.Biophys.
Acta,
I 16 (1966) 181-183
PRELIMINARY
NOTES
183
is derived from linoleic acid g. It is not possible from these results alone to deduce with certainty the structure of the other isomer, 20: 3 (I), but comparison of the “equivalent chain lengths” of the z isomers with the tabulated values given by HOFSTETTER et al.10 indicate that this acid is A VTll-eicosatrienoic acid which is known to be derived from oleic acids. GIDEZ~’ has recently demonstrated the presence of 2 isomeric eicosatrienoic acids in the adrenal cholesterol esters of the rat. Tentative identification, based on gas-chromatographic retention properties only, suggested that I isomer, present in small amounts in normal rats, was probably As~l~14-eicosatrienoic acid, whereas the other isomer, present only in rats deficient in essential fatty acid was probably A578711-eicosatrienoicacid. RILEY I2 has also suggested on theoretical grounds that an eicosatrienoic acid present in human adrenal glands might be the 118,11,14isomer. As far as we are aware the results presented in this communication are the acid in more than first to show conclusively the presence of A 8>11>14-eicosatrienoic trace quantities in rabbit tissues. High levels of this acid have however been reported in the cholesterol esters of canine adrenals 8p13.The accumulation of this acid in the adrenal cholesterol esters of rabbits given diets containing high levels of linoleic acid is in marked contrast to the many reports of an accumulation of eicosatrienoic acid in various tissues of animals given essential fatty acid-deficient diets. In view of these findings a note of caution should perhaps be sounded against the unqualified use of the trienoic acid: tetraenoic acid ratio as a diagnostic test for essential fatty acid status 14,15,particularly in studies involving the rabbit. J. H. MOORE D. L. WILLIAMS
National Institute fey Research in Dairy&g, ShinJield, Reading (Great Britain) I L. SWELL AND C. origin, Academic, 2 J. H. MOORE AND 3 J. H. MOORE AND 4 R. E. DAILEY, L.
R. TREADWELL, in M. SANDLER AND G. H. BOURNE, Athwosclerosis and its New York, 1963, p. 301. D. L. WILLIAMS, Ca?z. J. Biochenz. Physiol., qr (1963) 1821. D. L. WILLIAMS, B&t. J. N&r., 18 (1964) 603. SWELL, H. FIELD AND C. R. TREADWELL, PYOC. Sm. Exptl. Biol. Med., 105
(1960) 4. 5 J. H. MOORE AND D. L. WILLIAMS, Brit. J. N&r., 18 (1964) 253. 6 A. T. JAMES, J. Chvonzatog., z (1959) 552. 7 L. J. MORRIS, Chem. Ind. (Lo?zdon), (1962) 1238. 8 T. L. CHANG AND C. C. SWEELEY, r’. L&d Res., 3 (1962) 170. 9 J. F. MEAD, in K. BLOCH, Lipid A?etab&sm, Wile<, New York, 1960, p. 41. IO H. H. HOFSTETTER, N. SEN AND R. T. HOLMAN, I. Am. Oil Chavnists’ Sac.. 42 (1965) 577. , _ -, --, I I L. I. GIDEZ, Biochem. Biophys. Res. Commun., 14~(1964) 413. 12 C. RILEY, Biochem. J., 87 (7963) 500. 13 T. L. CHANG AND C. C. SWEELEY, Biochemistry, 2 (1963) 592. 14 H. F. WIESE, A. E. HANSEN AND D. J. D. ADAM, J. Nutv., 66 (1958) 345. 15 R. T. HOLMAN, J. N&r., 70 (1960) 405.
Received August 4th, 1965. Biochivn.
Biophys.
Acta,
116 (1966) 181-183