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Formula α-linolenic (18:3 (n−3)) and linoleic (18:2(n−6)) acid influence neonatal piglet liver and brain saturated fatty acids, as well as docosahexaenoic acid (22:6 (n−3))
262
BBALIP
53898
Formula cu-linolenic ( 18 : 3 ( n - 3)) and linoleic ( 18 : 2 ( n - 6) 1 acid influence neonatal piglet liver and brain saturated fatty acids, as well as docosahexaenoic acid (22 : 6 ( n - 3) 1 L. Dianne
Arbuckle
‘, France M. Rioux ‘, Murray J. MacKinnon and Sheila M. Innis ’
”
Departments ofu Human Nutrition and ’ Pediatrics, University of British Columbiu, Vancortr’rr (Canada) and ’ Statistical Consulting Sewice, The Childrenk Hospital, Vancotclser (Cunadal (Received
Key words:
Palmitic
4 October
1991)
acid: Essential fatty acid; Neonatal piglet: (Pig brain): (Pig liver)
Saturated fatty acids can be synthesized de novo and play a role in determining properties of structural membranes. The effect of dietary essential fatty acids, linoleic acid (18 : 2 (n - 6)) and a-linolenic acid (18 : 3 (n - 3)), on the saturated fatty acid content of membrane phospholipid has not previously been considered in newborn nutrition. The studies report the effect of low (1% fatty acids) or high (4%) formula 18:3 (n - 3) with low (16%) or high (30-35%) formula 18:2 (n - 6) on the saturated and unsaturated fatty acid composition of liver and brain structural lipid of piglets fed formula from birth for 15 days. A significant inverse relationship between the formula % 18:3 (n - 3), but not 18:2 (n - 6), and the liver phospholipid palmitic acid (16:O) was found. This may indicate a possible effect of dietary 18: 3 (12 - 3) on de novo synthesis of 16: 0 and requires further investigation. Monounsaturated fatty acids in both liver and brain were significantly lower in response to high 18 : 3 (n -- 3) and to high 18 : 2 (n - 6) plus low 18 : 1 (n - 9) in the formula. Liver phospholipid and brain total lipid % docosahexaenoic acid (22 : 6 (n - 3)) were significantly higher when formula containing 4% rather than 1% 18: 3 (n - 3) was fed, suggesting that 1% 18:3 (n - 3) may limit tissue (n - 3) fatty acid accretion. These results suggest that future studies of essential fatty acid requirements. specifically 18 : 3 (n - 3), should consider possible influences on the saturated fatty acids which also play a functional role in tissue structural lipids.
Introduction Fat represents 40 to 50% of the total kilocalories in breast milk and infant formulas. In addition to being the major energy source, the quality of the diet lipid is important because it supplies essential fatty acids crucial to normal growth of new tissue membrane lipids. The physical properties and biochemical functions of cell membrane structural phospholipids are determined, in part, by carbon chain length and degree of unsaturation of the constituent fatty acids [1,2]. In most phospholipids, the sn-1 position is usually occupied by a saturated fatty acyl chain, and the sn-2 position by an unsaturated fatty acyl chain [l]. The saturated fatty acids, which are predominantly palmitic (16: 0) and
Correspondence: Research Centre, V5Z 4H4.
S.M. Innis, Department of Pediatrics, 179-950 W. 28th Avenue, Vancouver,
U.B.C.. The B.C., Canada
stearic (18 :O) acid, decrease membrane lipid fluidity. Thus, the proportion of saturated fatty acids in the membrane lipid tends to be inversely related to membrane fluidity [ 1,2]. Animal studies have indicated that the diet fat content and quality can influence rates of de novo fatty acid synthesis [31. The possible importance of dietary saturated fatty acids to the desaturation and elongation products of the dietary essential fatty acids, linoleic (18 : 2 (n - 6)) and a-linolenic (18 : 3 (n - 3)) acid, in tissue lipids has been reported [4,5]. The effect of the quantity of 18: 2 (n - 6) and 18: 3 (n - 3) in infant diets on the composition of saturated fatty acids in structural lipids, however, has not been specifically studied. The purpose of this report is to describe the effect of high and low levels of 18 : 2 (n - 6) and/or 18 : 3 (n - 3) in infant formula diets on the composition of saturated, as well as 20 and 22-carbon long-chain polyunsaturated fatty acids, in the structural lipid of neonatal piglet liver and brain. The usefulness of the
263 piglet for study of infant formula containing about 50% kCa from fat during the normal suckling period, as it pertains to the human infant, has been discussed [6]. Materials and Methods
tion of the four formulas is in Table I. After opening, the liquid, ready-to-feed formulas were kept at 4°C for no longer than 24 h. The procedures used were approved by the University of British Columbia Animal Care Committee and conformed to the guidelines of the Canadian Council on Animal Care.
Animals and diets
Male Yorkshire piglets of normal term gestation (116-118 days) were obtained from the University of British Columbia, Department of Animal Science and Pitt Ineffable Growers (Pitt Meadows, B.C.). Six nonlittermate piglets for each of the four formula groups were taken from the sow immediately post partum, prior to receiving colostrum. Housing, feeding and provision of passive immunity with pig serum-derived immupoglobulin have been described previously [6]. All animals were fed from birth to 15 days of age, representing at least two-thirds of the normal suckling period. The macronutrient composition of the formula was based on that of infant formula, but modified to resemble the composition of sow milk and to meet National Research Council requirements for the growing pig [7]. The formula fatty acids differed in % fatty acids 18 : 2 (n - 6) and 18 : 3 (n - 3) to give low (16%) or high (30 or 35%) 18: 2 (n - 6) and low (1%) or high (4%) 18 : 3 (n - 3). Each level of 18 : 2 (n - 6) was fed with low (1%) or high (4%) 18: 3 (n - 3) (i.e., % 18: 2 (n - 6)/% 18: 3 (n - 3): 16/l, 16/4, 30/l and 35/4) to allow study of possible independent and interactive effects of formula fat content of 18 : 2 (n - 6) and 18 : 3 (n - 3). The manipulation of the formula 18 : 2 (n - 6) resulted in lower % 18: 1 (n - 9) in the high than low 18 : 2 (n - 6) formulas. There was a lower % fatty acids of carbon chain s 14 in the 4% than 1% 18: 3 (n - 3) formulas. Other fatty acid components were similar among the diets. The fat blend and fatty acid composi-
TABLE
I
Fatty acid composition
(% total fatty acids) of formulas
Differing formula % 18 : 2 (n - 6) and 18: 3 (n - 3) were achieved by various vegetable oil blends: 16/l, corn, soy and high-oleic sunflower; 16/4, soy, canola and high oleic-sunflower; 30/l, corn; 35/4, soy. All formulas contained 40% coconut oil as the source of saturated fatty acid. Fatty acid composition was determined by direct 1 h transesterification WO”C) with methanol-benzene (4: 1 v/v) and acetyl chloride [ll] and subsequent analysis by gas liquid chromatography
161.
Fatty acid
d 14:o 16:0 18:0 18:l (n -9) 18:2(n-6) 18:3(n--3)
%18:2(n-6)/%18:3(n-3) 16/l
16/4
30/l
35/4
32.2 8.1 3.4 38.6 15.6 0.7
28.3 7.9 3.8 40.1 16.6 3.9
38.9 10.4 2.8 17.3 29.5 0.8
27.5 10.4 4.6 17.2 35.1 4.5
Sample collection and analyses
The piglets were killed 15 days after birth by intracardiac injection of 10 ml KC1 (20 mequiv./lO ml). The cerebrum was immediately excised, weighed, minced and homogenized in 5 vol./wt. of 0.32 M sucrose-15 mM Tris-HCl: 1 mM EDTA, 1 mM MgCl, and 1.5 mM glutathione (pH 7.4). The liver was removed, weighed and homogenized in 50 ml of 0.9% sodium chloride plus 1.5% EDTA. Aliquots of brain and liver homogenate were stored at -80°C until analysis. Lipid and fatty acid analysis
Total lipids were extracted from brain and liver according to Folch et al. [8]. Liver total phospholipid (PL) was separated from other lipid classes by thin-layer chromatography [9]. Fatty acid methyl esters of brain total fatty acid (TFA) and liver PL were prepared, then separated, identified and quantified by gas liquid chromatography [6]. Statistical analysis
Two-way ANOVA [lo] was used to examine the effect of varying levels of 18 : 2 (n - 6) and 18 : 3 (n - 3) in the formula, and their interaction with respect to tissue fatty acid response. The level of significance was set at P < 0.05. Where an interaction between 18: 2 (n - 6) and 18: 3 (n - 3) was found to influence the fatty acid response, post hoc contrasts (Fisher’s least significant difference) were used for specific study of the effect of 18 : 3 (n - 3) level in the low or high 18 : 2 (n - 6) formulas. These formal tests of differences were based on least squares means and standard errors calculated from the ANOVA. Bonferroni corrections within a fatty acid were used to determine which of these differences were significant (P < 0.01). All calculations were performed using the General Linear Models procedure in the Number Cruncher Statistical System, version 5.01 (Kaysville, UT). Results and Discussion The results of this study show a significant inverse relationship between the level of 18: 3 (n - 3), but not 18: 2 (n - 61, in formula and the liver PL % 16: 0 (Table II). This is clearly illustrated (Fig. la) in that within each level of 18 : 2 (n - 61, increasing 18 : 3 (n 3) in the formula from 1 to 4% of fatty acids resulted in lower liver PL 16: 0. The response of brain TFA % 16 : 0 to diet was different in that it was dependent on
264 the diet 18: 2 (n - 6), as well as 18: 3 (n - 3) (Table III, Fig. lb). Results of the 2-way ANOVA on the effect of formula 18:2 (n - 6) and 18: 3 (n - 3) show that 16: 0 in the brain was significantly lower in piglets fed 30-35% compared to 16% 18: 2 (n - 6), and, as in the liver, was significantly lower in piglets fed 4% rather than 1% 18: 3 (n ~ 3). In contrast to the effect on 16:0, Fig. 1 a,b shows that the liver PL and brain TFA % 22 : 6 (n - 3) was significantly higher when the formula with 4 rather than 1% 18:3 (n -- 3) (1.7 and 0.3% kCa, respectively) was fed. This result suggests that the formulas with 1% 18 : 3 (n - 3) were limiting in (n - 3) fatty acids. Similar findings for centraI nervous tissue membranes of piglets [6,12-141, rodents [X-17] and monkeys [18] fed diets varying in 18 : 3 (n - 3) have been published. The amount of 18: 2 (17 - 6) in the formula fatty acids had no significant effect on the brain or liver 22 : 6 (n - 3) (Tables II, III). The possible relationship between the higher tissue 22: 6 (n - 3) and lower 16: 0 in the piglets fed 4% rather than 1% 18:3 (n - 3) must be considered. Any
TABLE
postulated biochemical mechanism, however, must be compatible with the finding that thcsc changes were more predominant in the liver than brain. Brain levels of triacylglycerol and cholesterol esters are very 1~ and the lipid present is predominantly structural 1191. The analyses of brain, however, included the structural lipid fatty acids of myelin which contain large amounts of saturated and monounsaturated fatty acids [I Y,20]. Analyses of brain TFA in growing piglets, therefore, reflects the developmental increase in the relative amount of myelin to the neural mcmbrancs, as well as maturation or diet-modifications of their fatty acid composition, The extent to which this rather than differences in fatty acid metabolism contributes to I he differing response of brain TFA compared to liver PL is unclear. The brain has the capacity for de nova synthesis of saturated and monounsaturated fatty acids [19], but also takes up these fatty acids from the plasma [21]. The relative importance of uptake from plasma compared to in situ synthesis to the incorporation 01 saturated and monounsaturated fatty acids into the brain is not known.
II and resuits of 2-way ANOVA f or significant differences due to jbrmula content o,f 18: 2 (11~ 61 untl ih’:.?
f.icvr phospholipid futty acid composition (n - 31
Data represent mean + S.E. in = 6). Where P-value indicates a significant group comparisons, determined using Fisher’s least significant difference,
interactive effect of formula 18: i7 (n with Bonferroni correction. are given.
16
c/n 18:3(n-3)
1
and 18: 3 (n - 3). individual
P-value
% of total fatty acids $6 I8:2 (n -6)
6)
1x:2 (n -6)
> 30 4
1
lK:3(n-3)
interaction
< O.OOtIl NS <0.0001
NS NS NS
c 0.000I 0.03 O.OOOK
0.0000 0.05 0.003
4
15.5 + 0.6 28.4 + 0.5 45.7 f 0.4
_ ‘NS NS 0.05
12.2*0.3 2x.3-I_0.7 42.2+0.5
16:0 1x:0 ‘.ZSats
15.9+0.5 27.8k 1.1 44.7 + 0.8
11.8+0.3 27.5 + 0.3 41.1+0.4
1h:l (n-7) 18:l (n-9) “1’Monos
0.3 f 0.0 i.d 15.2 kO.6 16.4 +O.h
(1.I + 0.0 13.7 2 0.2 “ 14.or 0.2 iI
IX:2 C/l-6)
1fJ.4+0.7 17.lkO.6 1x.8+0.9
16.5 i 11.3 17.1+0.3
22.2 * 0.2 18.8 f 0.5 21.5 kO.5
22.5 -f 0.X 17.6; 0.5 18.4iO.S
< 0.000 f ll.1)1 0.002
0.1 + 0.0 2.0 * 0.3 2.5 * 0.4
0.3 i 0.0 7.2 i 0.3 9.4 * 0.2
0.1 +o.o 1.5tn.1 1.9*0.1
0.210.1 6.5 i 0.5 8.4 + 0,s
NS NS OS)2
O.Oil5
21.3* 1.2 19.4+0.1 142.1 15.0
26.4 * 0.4 19.8+0.0 174.3+ 1.1
26.8 * 0.9 19.7 * 0.0 17x.4 + 3.0
NS NS OK?
< 0.0001 0.0001 < 0.000 t
20:4(n-6) -iz’ t II - 6)LCP 18:3 (n -3) 22:6 (n -3) hz (n - 3)LCP ‘2‘LCP “MChL ‘UI
I h.S- 0.4
0.I f 0.0
0.1t 0.0
6.7kO.l i7.11+0.1 ”
h.h+o.2 ” 6.82 0.2 j’
23.4 + 0.5 lY.6+0.0 154.0 + 1.6
’ NS, not statistically significant. ’ TSats, sum of 16:0, 18:0, 20:0, 22:0 and 24:0. ’ Values in the same row with different superscript letter are significantly different ’ PMonos, sum of 16: 1 (n -7). 18: 1 (n -9), 20: 1 (n -9) and 22:l (II -9). 5 2’(n~6)LCP,sumof20:4(n~6),22:4(n-6)~lnd22:5(rl-6). h X(n -3)LCP, sum of 20:5 (n -3) 22:5 tn -3) and 22:h (n-3). ’ ,CLCP, sum of (n - 6) and (n - 3) LCP. ’ MChL, mean chain length. ‘I UI, unsaturation index = 2 (No. double bonds X % fatty acid)
< 0.000 1 < 0.000 1 < 11.000 1
(P < 0.01).
NS
NS
NS O.OOO5
NS NS NS NS NS NS NS
NS
265 petition for malonyl-CoA between de novo synthesis of 16 : 0 and elongation of 18 : 3 (n - 3) during metabolism to 22 : 6 (n - 3) may favor 18 : 3 (n - 3). Possible support for this theory is provided by results showing that the decrease in liver PL % 16 :0 was of a similar magnitude to the increase in % 22 : 6 (n - 3). The fatty acid composition of phospholipids is the result not only of fatty acids available from synthesis or the diet, but aIso reflects the specificities of the acyltransferase involved with de novo synthesis or remodelling. Thus, rather than changes in 16: 0 synthesis, differences in phospholipid content of 16: 0 could be due to differences in incorporation of 16: 0 during phospholipid synthesis and remodelling. It seems unlikely that the decreased liver PL % 16 : 0 in piglets fed 4% compared to 1% 18 :3 (n - 3) (Table II) is explained by competition between 16 : 0 and 22 : 6 (n - 3) for acylation because acylation of saturated and polyunsaturated fatty acids is usually at the sn-1 and ~2-2 position of phospholipid, respectively [l]. The results also offer no explanation of the decrease in liver PL 16: 0 due to increased acylation of some other fatty acid,suchas18:0or18:1(n-9),as22:6(n-3)was the only fatty acid significantly increased in piglets fed the high 18: 3 (n - 3) formula. Another possible reason for the decrease in 16 :0 is increased elongation and/or desaturation to 18:O or 16 : 1 (n - 7), respectively. As previously discussed, the tissue fatty acid analysis did not find increased levels of
The decreased tissue % 16:0 in the piglets fed the formula with the high 18 : 3 (n - 3) could possibly be related to decreased synthesis and/or increased utilization of 16 : 0. The formulas all contained similar low levels of 16 : 0 and 18 : 0 (8 and 4% of total fatty acids, respectively), suggesting that the high levels of these fatty acids in phospholipids of brain and liver are likely to originate from de novo synthesis. The quantity of carbon chain 6 14 fatty acids was 5-10% lower in the formulas with 4% 18: 3 (n - 3) than in the formulas with 1% 18: 3 (n - 3) (Table I). Although these medium-chain fatty acids are known to be rapidly oxidized, the acetyl-CoA produced during oxidation may be a source of 2-carbon units for elongation of endogenous fatty acids, or elongation to 16: 0 could occur [22] and thus explain the differences found in the tissue 16 :0. De novo synthesis of 16 :0 is known to be inhibited by 18 : 3 (n - 3), both by short-term inhibition of acetyl CoA carboxylase [23] and by a long-term adaptive response in which the activity and synthesis of acetyl CoA carboxylase [24,25] and fatty acid synthetase [24271 are decreased. Furthermore, the malonyl CoA used for elongation of fatty acids during synthesis in the cytosol is probably derived from the same pool providing 2-carbon units for desaturation/elongation of 18 : 3 (n - 3) in the microsome [28]. Microsomal elongation is more active with unsaturated than saturated fatty acid substrates [28]. It is possible, therefore, that com-
TABLE III Bruin total fatty acid composition and results of 2-way ANOVA for significant differences due to formula content of 18:2 (n - 6) and 18:3 (n - 3)
Data represent mean f SE. (n = 6). Where P-value indicates a significant interactive effect of formula 18 : 2 (n - 6) and 18 : 3 (n - 3), individual group comparisons, determined using Fisher’s least significant difference, with Bonferroni correction, are given. % of total fatty acids % 18:2(n-6)
16
% 18:3(n-3)
1
P-value 2 30
4
1
18:2(n-6)
18:3 (n -3)
interaction
4
16:0 18:0 ZSats
21.1 f0.5 22.8iO.3 46.6+0.5
19.6 + 0.2 23.6fO.l 45.7kO.2
19.0f0.4 23.4 i 0.1 45.OkO.3
18.7kO.4 24.2 f 0.2 45.6 + 0.4
0.0006 0.004 0.02
0.03 0.0006 NS
NS 1 NS NS
16:l(n-7) 18:1(n-9) Z Monos
2.0*0.1 20.4iO.2 24.6iO.2
1.7kO.l 19.7 f 0.3 23.7kO.4
1.5 kO.1 19.8+0.3 23.6 f 0.4
1.4iO.l 18.5kO.l 22.2io.2
0.0006 0.001 0.0004
0.05 0.0006 0.001
NS NS NS
18:2(n-6) 20:4(n-6) 2 (n - 6)LCP
1.5 io.0 10.3 f 0.3 16.5 f 0.7
1.6kO.O 10.3 * 0.2 16.2kO.2
1.8+0.1 ‘.a 11.2*0.2 19.0 + 0.2
2.3 + 0.1 b 10.6iO.2 17.0*0.3
< 0.0001 0.007 0.0004
< 0.0001 NS 0.008
0.002 NS 0.05
0.1 *o.o 8.8iO.l 9.3 +0.2
0.2*0.1 10.3 + 0.2 11.0*0.2
tr 8.6kO.2 8.9 & 0.2
0.1+ 0.0 10.3 f 0.3 11.0*0.3
0.05 NS NS
NS < 0.0001 < 0.0001
NS NS NS
25.8*0.6 18.0k0.0 154.3 + 2.4
27.2 f 0.3 18.0 f 0.0 162.7 f 1.4
27.9 k 0.2 18.1+ 0.0 162.7 f 0.7
28.0 f 0.4 18.1 fO.O 166.0 f 1.8
0.002 NS 0.002
NS NS 0.002
NS NS NS
18:3 (n -3) 22:6(n-3)
,z (n - 3)LCP PLCP MChL UI
’ See footnote Table II for abbreviations ’ Values in the same row with different superscript letter are significantly different (P < 0.01).
Fig. I. Plots to show 16:0,
1X: 1, 1X:.! (n -6)
and 22:6
total fatty acids) in: (a) liver total phospholipid: fatty 16/4;
acid of piglets hatched,
30/l;
fed formula
containing:
and black. 35/4
YIX:1
(n -3)
(5
and (b) brain total
crossed. (II -6)/“ilX:3
16/l:
clear, (II -3)
are given in Tables II and III.
these fatty acids in piglets fed the high 18:3 (n - 3) formulas. In fact, liver PL and brain levels of 16: 1 (n - 7) and 18: 1 (n - 9) were lower in piglets fed 4% compared to 1% 18: 3 (n - 3) in formula (Tables II, III). Possibly this could be explained by inhibition of A”-desaturase by 18: 3 (n - 3) [291. Significant changes in the 2 monounsaturated fatty acids, predominantly 18: 1 (n ~ 9), of brain and liver were seen in response to formula 18 : 2 (n - 6). The changes attributed to formula 18: 2 (n - 6), however, are probably most appropriately discussed with respect to formula content of 18: 1 (n - 9). Liver PL and brain TFA 18: 1 (n - 9) were about 50% and 5% lower, respectively, when formula containing high 18 : 2 (n - 6) (low 18: 1 (n - 9)) rather than low 18: 2 (n - 6) (high 18: 1 (n - 9)) was fed (Tables II, III; Fig. 1 a,b). Possibly, the more limited effect of the formula 18 : 1 (n - 9) and 18: 2 (n - 6) content on brain than liver reflects de novo fatty acid synthesis by the oligodendrocytes which are known to synthesize large amounts of myelin lipid, containing particularly high 18: 1 (n - 9) [19,20], during the first 3 weeks after birth in the piglet [32]. Levels of 18 : 2 (n - 6) were increased from about
16 to 22% of total fatty acids in the liver PI.. but f’rom about 1.5 to 1.8-2.2s of fatty acids in the brain ot piglets fed the high compared to low IX : 2 (II 0) formulas. Thus, it may bc worth considering that the more prominent effects of the formula IX : 2 (II 6) Oil liver 18: 1 (n - 9) may also include the known inhibition of A”-desaturase by IX : 2 (II -- 6) [30,31]. As well as 18:2 (II - h), levels of 20:4 01 ~~~ 6) in the liver PL and brain TFA were incrcascd in response to high formula 18: 2 (II ~ 6) (Tables II. III: Fig. 1 a,h). The brain TFA Cs;,1X: 3 (II -~ 6) demonstrated a f’urthcl increase when 4% 18: 3 (n - 3) rather than 1’7 IX : 3 (n - 3) was fed with high It; : 2 (II .- 6). The unsaturation index of Iivcr Pt. and brain TFA showed independent, significant changes due to the formula 18 : 2 (n - 6) and 18 : 3 01 ~~3) content: a higher unsaturation index was assnciatcd with either high IX: 2 (n ~ 6) or high IX: 3 (12 ~ 3) in the formula (l’;tbles II, III). The change in unsaturation index was more pronounced in the liver than brain, and when the formula IX: 3 (n .- 3) rather than 1X: 2 (II 0) was increased (Table 11). The accompanying change in the liver PL mean chain length is explained by the combination of lower _“ saturated fatty acids and higher long-chain polyunsaturated fatty acids. primarily 27 : h (n - .i), when the high 1X :i (II ~~3) formula wax fed. Alterations in membrane protein activities have been associated with changes in the unsaturation index ol’. for example, total phospholipids of liver microsomal membrane [33] and plasma membrane [34]. Whethel the small, but statistically Ggnificant. change\ in liver fatty acid composition and membrane unsaturation seen in this study are of physiological relevance to the newborn is not known. In contrast to the tivcr. the highly significant (I’ < 0.000 1) incrcasc in brain 22 : h (II - 3) when 4 rather than l’i IX:3 (II 3) W;I~ fed. had no effect on the brain total long-chain polyunsaturated fatty acids or mean chain length. This i%prohablq cxplaincd by the well known rcplaccmcnt of 21: 0 (?I - 3) with 22: 4 (II -. 6) and particular’ll, 37 : 5 (or 6) in central nervous system lipids of (II 3) t’atty acid-dcficient animals [ 15- IS]. This study has demonstrated an inverse relationship between formula 1X:3 (II ~~1) and the c; 16: 0 in structural phospholipid of the liver. A similar relationship occurs in the brain when high IX : 3 (II - 3i is fed with 16% 18: 2 (II ~ 0). This is. to our knowlcdgc, the first report of a potential interaction between the dietary intake of 1X: 3 (II - 3) and deposition of 16 : 0 in growing tissues. The possible mechanisms responsible arc speculative and cannot he determined from the results here. Further studies are required to investigate the effect of formula 18: 3 (II - 3) on dc nova saturated fatty acid synthesis. The effect ~1 dictar) medium-chain fatty acids on the synthesis or elongation of fatty acids also requires investigation. In conclu-
267 sion, determination of essential fatty acid requirements, specifically 18 : 3 (n - 31, should consider possible influences on the non-essential fatty acids which also play a role in determining the properties of structural membranes of important organs such as the liver. Acknowledgments
This work was supported by grants from the Medical Research Council of Canada (MRC-Industry Award) and the Canola Utilization Assistance Program. The formulas were prepared and generously donated by Ross Laboratories, Columbus, Ohio. L.D.A. is a recipient of the 1967 Science and Engineering Scholarship, Natural Sciences and Engineering Research Council of Canada. S.M.I. is a Career Investigator of the British Columbia’s Children’s Hospital Foundation. References 1 Stubbs, C.D. and Smith, A.D. (1984) Biochim. Biophys. Acta 779,
89-137. 2 McMurchie, E.J. (1988) in Physiological Regulation of Membrane Fluidity (Aloia, R.C., Curtain, C.C. and Gordon, L.M., eds.), pp. 189-237, Alan R. Liss, New York. Herzberg, G.R. (1983) Adv. Nutr. Res. 5, 221-253. Garg, M.L., Wierzbicki, A.A., Thomson, A.B.R. and Clandinin, M.T. (1989) Lipids 24, 334-339. Brenner, R.R. and Peluffo, R.O. (1966) J. Biol. Chem. 241, 5213-5219. Hrboticky, N., MacKinnon, M.J. Puterman, M.L. and Innis, S.M. (1989) J. Lipid Res. 30, 1173-1184. NRC, Sub-Committee on Swine Nutrition. (1979) in Nutrient Requirements of Swine, 8th Edn., pp. 22-23, National Academic Press, Washington, D.C. 8 Folch, J., Lees, M. and Stanley, G.H.S. (1957) J. Biol. Chem. 226, 497-509. 9 Innis, S.M. and Clandinin, M.T. (1981) Biochem. J. 193, 155-167. 10 Montgomery, D.C. (1984) Design and Analysis of Experiments, 2nd Edn., pp. 43-121, John Wiley & Sons, New York.
11 Lepage, G. and Roy, C.C. (1986) J. Lipid Res. 27, 114-120. 12 Arbuckle, L.D. and Innis, S.M. (1991) Lipids, in press. 13 Hrboticky, N., MacKinnon, M.J. and Innis, S.M. (1990) Am. J. Clin. Nutr. 51, 173-182. 14 Hrboticky, N., MacKinnon, M.J. and Innis, S.M. (1991) Am. J. Clin. Nutr. 53, 483-490. 15 Bourre, J.M., Francois, M., Youyou, A., Dumont, O., Piciotti, M., Pascal, _G. and Durand, G. (1989) J. Nutr. 119, 1880-1892. 16 Anding, R.H. and Hwang, D.H. (1986) Lipids 21, 697-701. 17 Yamamoto, N., Sitoh, M, Moriuchi, A. and Nomura, M. (1987) J. Lipid Res. 28, 144-151. 18 Connor, W.E. and Neuringer, M. (1988) in Biological Membranes: Aberrations in Membrane Structure and Function, pp. 275-294, Alan R. Liss, New York 19 Sastry, P.S. (1985) Prog. Lipid Res. 24, 69-176. 20 Norton, W.T. and Cammer, W. (1984) in Myelin, 2nd Edn., (Morell, P., ed.), pp. 147-198, Plenum Press, New York. 21 Dhopeshwarkar, G.A. (1973) Adv. Lipid Res. 11, 109-142. 22 Bach, A.C. and Babayan, V.K. (1982) Am. J. Clin. Nutr. 36, 950-962. 23 Bloch, K. and Vance, D. (1977) Annu. Rev. Biochem. 46,283-298. 24 Toussant, M.J., Wilson, M.D. and Clarke, S.D. (1981) J. Nutr. 111, 146-153. 25 Abraham, S., Hillyard, L.A., Lin, C.Y. and Schwartz. R.S. (1983) Lipids 18, 820-829. 26 Clarke, S.D., Romsos, D.R. and Leveille, G.A. (1977) J. Nutr. 107, 1170-1181. 27 Schwartz, R.S. and Abraham, S. (1982) Biochim. Biophys. Acta 711, 316-326. 28 Cook, H.W. (1985) in Biochemistry of Lipids and Membranes (Vance, D.E. and Vance, J.E., eds.), pp. 181-212, The Benjamin/Cummings Publishing Company, Menlo Park, CA. 29 Mahfouz, M.M., Smith, T.L. and Kummerow, F.A. (1984) Lipids 19, 214-222. 30 Garg, M.L., Wierzbicki, A.A., Thomson, A.B.R. and Clandinin, M.T. (1988) Biochim. Biophys. Acta 962, 330-336. 31 Garg, M.L., Snoswell, A.M. and Sabine, J.R. (1986) Prog. Lipid Res. 25, 639-644. 32 Sweasy, D., Patterson, D.S.P. and Glancy, E.M. (1976) J. Neurochem. 27, 375-380. 33 Christon, R., Fernandez, Y., Cambon-Gros, C., Periquet, A., Deltour, P., Leger, CL. and Mitjavila, S. (1988) J. Nutr. 118, 1311-1318. 34 Morson, L.A. and Clandinin, M.T. (1986) J. Nutr. 116,2355-2362.
BBALIP
53898
Formula cu-linolenic ( 18 : 3 ( n - 3)) and linoleic ( 18 : 2 ( n - 6) 1 acid influence neonatal piglet liver and brain saturated fatty acids, as well as docosahexaenoic acid (22 : 6 ( n - 3) 1 L. Dianne
Arbuckle
‘, France M. Rioux ‘, Murray J. MacKinnon and Sheila M. Innis ’
”
Departments ofu Human Nutrition and ’ Pediatrics, University of British Columbiu, Vancortr’rr (Canada) and ’ Statistical Consulting Sewice, The Childrenk Hospital, Vancotclser (Cunadal (Received
Key words:
Palmitic
4 October
1991)
acid: Essential fatty acid; Neonatal piglet: (Pig brain): (Pig liver)
Saturated fatty acids can be synthesized de novo and play a role in determining properties of structural membranes. The effect of dietary essential fatty acids, linoleic acid (18 : 2 (n - 6)) and a-linolenic acid (18 : 3 (n - 3)), on the saturated fatty acid content of membrane phospholipid has not previously been considered in newborn nutrition. The studies report the effect of low (1% fatty acids) or high (4%) formula 18:3 (n - 3) with low (16%) or high (30-35%) formula 18:2 (n - 6) on the saturated and unsaturated fatty acid composition of liver and brain structural lipid of piglets fed formula from birth for 15 days. A significant inverse relationship between the formula % 18:3 (n - 3), but not 18:2 (n - 6), and the liver phospholipid palmitic acid (16:O) was found. This may indicate a possible effect of dietary 18: 3 (12 - 3) on de novo synthesis of 16: 0 and requires further investigation. Monounsaturated fatty acids in both liver and brain were significantly lower in response to high 18 : 3 (n -- 3) and to high 18 : 2 (n - 6) plus low 18 : 1 (n - 9) in the formula. Liver phospholipid and brain total lipid % docosahexaenoic acid (22 : 6 (n - 3)) were significantly higher when formula containing 4% rather than 1% 18: 3 (n - 3) was fed, suggesting that 1% 18:3 (n - 3) may limit tissue (n - 3) fatty acid accretion. These results suggest that future studies of essential fatty acid requirements. specifically 18 : 3 (n - 3), should consider possible influences on the saturated fatty acids which also play a functional role in tissue structural lipids.
Introduction Fat represents 40 to 50% of the total kilocalories in breast milk and infant formulas. In addition to being the major energy source, the quality of the diet lipid is important because it supplies essential fatty acids crucial to normal growth of new tissue membrane lipids. The physical properties and biochemical functions of cell membrane structural phospholipids are determined, in part, by carbon chain length and degree of unsaturation of the constituent fatty acids [1,2]. In most phospholipids, the sn-1 position is usually occupied by a saturated fatty acyl chain, and the sn-2 position by an unsaturated fatty acyl chain [l]. The saturated fatty acids, which are predominantly palmitic (16: 0) and
Correspondence: Research Centre, V5Z 4H4.
S.M. Innis, Department of Pediatrics, 179-950 W. 28th Avenue, Vancouver,
U.B.C.. The B.C., Canada
stearic (18 :O) acid, decrease membrane lipid fluidity. Thus, the proportion of saturated fatty acids in the membrane lipid tends to be inversely related to membrane fluidity [ 1,2]. Animal studies have indicated that the diet fat content and quality can influence rates of de novo fatty acid synthesis [31. The possible importance of dietary saturated fatty acids to the desaturation and elongation products of the dietary essential fatty acids, linoleic (18 : 2 (n - 6)) and a-linolenic (18 : 3 (n - 3)) acid, in tissue lipids has been reported [4,5]. The effect of the quantity of 18: 2 (n - 6) and 18: 3 (n - 3) in infant diets on the composition of saturated fatty acids in structural lipids, however, has not been specifically studied. The purpose of this report is to describe the effect of high and low levels of 18 : 2 (n - 6) and/or 18 : 3 (n - 3) in infant formula diets on the composition of saturated, as well as 20 and 22-carbon long-chain polyunsaturated fatty acids, in the structural lipid of neonatal piglet liver and brain. The usefulness of the
263 piglet for study of infant formula containing about 50% kCa from fat during the normal suckling period, as it pertains to the human infant, has been discussed [6]. Materials and Methods
tion of the four formulas is in Table I. After opening, the liquid, ready-to-feed formulas were kept at 4°C for no longer than 24 h. The procedures used were approved by the University of British Columbia Animal Care Committee and conformed to the guidelines of the Canadian Council on Animal Care.
Animals and diets
Male Yorkshire piglets of normal term gestation (116-118 days) were obtained from the University of British Columbia, Department of Animal Science and Pitt Ineffable Growers (Pitt Meadows, B.C.). Six nonlittermate piglets for each of the four formula groups were taken from the sow immediately post partum, prior to receiving colostrum. Housing, feeding and provision of passive immunity with pig serum-derived immupoglobulin have been described previously [6]. All animals were fed from birth to 15 days of age, representing at least two-thirds of the normal suckling period. The macronutrient composition of the formula was based on that of infant formula, but modified to resemble the composition of sow milk and to meet National Research Council requirements for the growing pig [7]. The formula fatty acids differed in % fatty acids 18 : 2 (n - 6) and 18 : 3 (n - 3) to give low (16%) or high (30 or 35%) 18: 2 (n - 6) and low (1%) or high (4%) 18 : 3 (n - 3). Each level of 18 : 2 (n - 6) was fed with low (1%) or high (4%) 18: 3 (n - 3) (i.e., % 18: 2 (n - 6)/% 18: 3 (n - 3): 16/l, 16/4, 30/l and 35/4) to allow study of possible independent and interactive effects of formula fat content of 18 : 2 (n - 6) and 18 : 3 (n - 3). The manipulation of the formula 18 : 2 (n - 6) resulted in lower % 18: 1 (n - 9) in the high than low 18 : 2 (n - 6) formulas. There was a lower % fatty acids of carbon chain s 14 in the 4% than 1% 18: 3 (n - 3) formulas. Other fatty acid components were similar among the diets. The fat blend and fatty acid composi-
TABLE
I
Fatty acid composition
(% total fatty acids) of formulas
Differing formula % 18 : 2 (n - 6) and 18: 3 (n - 3) were achieved by various vegetable oil blends: 16/l, corn, soy and high-oleic sunflower; 16/4, soy, canola and high oleic-sunflower; 30/l, corn; 35/4, soy. All formulas contained 40% coconut oil as the source of saturated fatty acid. Fatty acid composition was determined by direct 1 h transesterification WO”C) with methanol-benzene (4: 1 v/v) and acetyl chloride [ll] and subsequent analysis by gas liquid chromatography
161.
Fatty acid
d 14:o 16:0 18:0 18:l (n -9) 18:2(n-6) 18:3(n--3)
%18:2(n-6)/%18:3(n-3) 16/l
16/4
30/l
35/4
32.2 8.1 3.4 38.6 15.6 0.7
28.3 7.9 3.8 40.1 16.6 3.9
38.9 10.4 2.8 17.3 29.5 0.8
27.5 10.4 4.6 17.2 35.1 4.5
Sample collection and analyses
The piglets were killed 15 days after birth by intracardiac injection of 10 ml KC1 (20 mequiv./lO ml). The cerebrum was immediately excised, weighed, minced and homogenized in 5 vol./wt. of 0.32 M sucrose-15 mM Tris-HCl: 1 mM EDTA, 1 mM MgCl, and 1.5 mM glutathione (pH 7.4). The liver was removed, weighed and homogenized in 50 ml of 0.9% sodium chloride plus 1.5% EDTA. Aliquots of brain and liver homogenate were stored at -80°C until analysis. Lipid and fatty acid analysis
Total lipids were extracted from brain and liver according to Folch et al. [8]. Liver total phospholipid (PL) was separated from other lipid classes by thin-layer chromatography [9]. Fatty acid methyl esters of brain total fatty acid (TFA) and liver PL were prepared, then separated, identified and quantified by gas liquid chromatography [6]. Statistical analysis
Two-way ANOVA [lo] was used to examine the effect of varying levels of 18 : 2 (n - 6) and 18 : 3 (n - 3) in the formula, and their interaction with respect to tissue fatty acid response. The level of significance was set at P < 0.05. Where an interaction between 18: 2 (n - 6) and 18: 3 (n - 3) was found to influence the fatty acid response, post hoc contrasts (Fisher’s least significant difference) were used for specific study of the effect of 18 : 3 (n - 3) level in the low or high 18 : 2 (n - 6) formulas. These formal tests of differences were based on least squares means and standard errors calculated from the ANOVA. Bonferroni corrections within a fatty acid were used to determine which of these differences were significant (P < 0.01). All calculations were performed using the General Linear Models procedure in the Number Cruncher Statistical System, version 5.01 (Kaysville, UT). Results and Discussion The results of this study show a significant inverse relationship between the level of 18: 3 (n - 3), but not 18: 2 (n - 61, in formula and the liver PL % 16: 0 (Table II). This is clearly illustrated (Fig. la) in that within each level of 18 : 2 (n - 61, increasing 18 : 3 (n 3) in the formula from 1 to 4% of fatty acids resulted in lower liver PL 16: 0. The response of brain TFA % 16 : 0 to diet was different in that it was dependent on
264 the diet 18: 2 (n - 6), as well as 18: 3 (n - 3) (Table III, Fig. lb). Results of the 2-way ANOVA on the effect of formula 18:2 (n - 6) and 18: 3 (n - 3) show that 16: 0 in the brain was significantly lower in piglets fed 30-35% compared to 16% 18: 2 (n - 6), and, as in the liver, was significantly lower in piglets fed 4% rather than 1% 18: 3 (n ~ 3). In contrast to the effect on 16:0, Fig. 1 a,b shows that the liver PL and brain TFA % 22 : 6 (n - 3) was significantly higher when the formula with 4 rather than 1% 18:3 (n -- 3) (1.7 and 0.3% kCa, respectively) was fed. This result suggests that the formulas with 1% 18 : 3 (n - 3) were limiting in (n - 3) fatty acids. Similar findings for centraI nervous tissue membranes of piglets [6,12-141, rodents [X-17] and monkeys [18] fed diets varying in 18 : 3 (n - 3) have been published. The amount of 18: 2 (17 - 6) in the formula fatty acids had no significant effect on the brain or liver 22 : 6 (n - 3) (Tables II, III). The possible relationship between the higher tissue 22: 6 (n - 3) and lower 16: 0 in the piglets fed 4% rather than 1% 18:3 (n - 3) must be considered. Any
TABLE
postulated biochemical mechanism, however, must be compatible with the finding that thcsc changes were more predominant in the liver than brain. Brain levels of triacylglycerol and cholesterol esters are very 1~ and the lipid present is predominantly structural 1191. The analyses of brain, however, included the structural lipid fatty acids of myelin which contain large amounts of saturated and monounsaturated fatty acids [I Y,20]. Analyses of brain TFA in growing piglets, therefore, reflects the developmental increase in the relative amount of myelin to the neural mcmbrancs, as well as maturation or diet-modifications of their fatty acid composition, The extent to which this rather than differences in fatty acid metabolism contributes to I he differing response of brain TFA compared to liver PL is unclear. The brain has the capacity for de nova synthesis of saturated and monounsaturated fatty acids [19], but also takes up these fatty acids from the plasma [21]. The relative importance of uptake from plasma compared to in situ synthesis to the incorporation 01 saturated and monounsaturated fatty acids into the brain is not known.
II and resuits of 2-way ANOVA f or significant differences due to jbrmula content o,f 18: 2 (11~ 61 untl ih’:.?
f.icvr phospholipid futty acid composition (n - 31
Data represent mean + S.E. in = 6). Where P-value indicates a significant group comparisons, determined using Fisher’s least significant difference,
interactive effect of formula 18: i7 (n with Bonferroni correction. are given.
16
c/n 18:3(n-3)
1
and 18: 3 (n - 3). individual
P-value
% of total fatty acids $6 I8:2 (n -6)
6)
1x:2 (n -6)
> 30 4
1
lK:3(n-3)
interaction
< O.OOtIl NS <0.0001
NS NS NS
c 0.000I 0.03 O.OOOK
0.0000 0.05 0.003
4
15.5 + 0.6 28.4 + 0.5 45.7 f 0.4
_ ‘NS NS 0.05
12.2*0.3 2x.3-I_0.7 42.2+0.5
16:0 1x:0 ‘.ZSats
15.9+0.5 27.8k 1.1 44.7 + 0.8
11.8+0.3 27.5 + 0.3 41.1+0.4
1h:l (n-7) 18:l (n-9) “1’Monos
0.3 f 0.0 i.d 15.2 kO.6 16.4 +O.h
(1.I + 0.0 13.7 2 0.2 “ 14.or 0.2 iI
IX:2 C/l-6)
1fJ.4+0.7 17.lkO.6 1x.8+0.9
16.5 i 11.3 17.1+0.3
22.2 * 0.2 18.8 f 0.5 21.5 kO.5
22.5 -f 0.X 17.6; 0.5 18.4iO.S
< 0.000 f ll.1)1 0.002
0.1 + 0.0 2.0 * 0.3 2.5 * 0.4
0.3 i 0.0 7.2 i 0.3 9.4 * 0.2
0.1 +o.o 1.5tn.1 1.9*0.1
0.210.1 6.5 i 0.5 8.4 + 0,s
NS NS OS)2
O.Oil5
21.3* 1.2 19.4+0.1 142.1 15.0
26.4 * 0.4 19.8+0.0 174.3+ 1.1
26.8 * 0.9 19.7 * 0.0 17x.4 + 3.0
NS NS OK?
< 0.0001 0.0001 < 0.000 t
20:4(n-6) -iz’ t II - 6)LCP 18:3 (n -3) 22:6 (n -3) hz (n - 3)LCP ‘2‘LCP “MChL ‘UI
I h.S- 0.4
0.I f 0.0
0.1t 0.0
6.7kO.l i7.11+0.1 ”
h.h+o.2 ” 6.82 0.2 j’
23.4 + 0.5 lY.6+0.0 154.0 + 1.6
’ NS, not statistically significant. ’ TSats, sum of 16:0, 18:0, 20:0, 22:0 and 24:0. ’ Values in the same row with different superscript letter are significantly different ’ PMonos, sum of 16: 1 (n -7). 18: 1 (n -9), 20: 1 (n -9) and 22:l (II -9). 5 2’(n~6)LCP,sumof20:4(n~6),22:4(n-6)~lnd22:5(rl-6). h X(n -3)LCP, sum of 20:5 (n -3) 22:5 tn -3) and 22:h (n-3). ’ ,CLCP, sum of (n - 6) and (n - 3) LCP. ’ MChL, mean chain length. ‘I UI, unsaturation index = 2 (No. double bonds X % fatty acid)
< 0.000 1 < 0.000 1 < 11.000 1
(P < 0.01).
NS
NS
NS O.OOO5
NS NS NS NS NS NS NS
NS
265 petition for malonyl-CoA between de novo synthesis of 16 : 0 and elongation of 18 : 3 (n - 3) during metabolism to 22 : 6 (n - 3) may favor 18 : 3 (n - 3). Possible support for this theory is provided by results showing that the decrease in liver PL % 16 :0 was of a similar magnitude to the increase in % 22 : 6 (n - 3). The fatty acid composition of phospholipids is the result not only of fatty acids available from synthesis or the diet, but aIso reflects the specificities of the acyltransferase involved with de novo synthesis or remodelling. Thus, rather than changes in 16: 0 synthesis, differences in phospholipid content of 16: 0 could be due to differences in incorporation of 16: 0 during phospholipid synthesis and remodelling. It seems unlikely that the decreased liver PL % 16 : 0 in piglets fed 4% compared to 1% 18 :3 (n - 3) (Table II) is explained by competition between 16 : 0 and 22 : 6 (n - 3) for acylation because acylation of saturated and polyunsaturated fatty acids is usually at the sn-1 and ~2-2 position of phospholipid, respectively [l]. The results also offer no explanation of the decrease in liver PL 16: 0 due to increased acylation of some other fatty acid,suchas18:0or18:1(n-9),as22:6(n-3)was the only fatty acid significantly increased in piglets fed the high 18: 3 (n - 3) formula. Another possible reason for the decrease in 16 :0 is increased elongation and/or desaturation to 18:O or 16 : 1 (n - 7), respectively. As previously discussed, the tissue fatty acid analysis did not find increased levels of
The decreased tissue % 16:0 in the piglets fed the formula with the high 18 : 3 (n - 3) could possibly be related to decreased synthesis and/or increased utilization of 16 : 0. The formulas all contained similar low levels of 16 : 0 and 18 : 0 (8 and 4% of total fatty acids, respectively), suggesting that the high levels of these fatty acids in phospholipids of brain and liver are likely to originate from de novo synthesis. The quantity of carbon chain 6 14 fatty acids was 5-10% lower in the formulas with 4% 18: 3 (n - 3) than in the formulas with 1% 18: 3 (n - 3) (Table I). Although these medium-chain fatty acids are known to be rapidly oxidized, the acetyl-CoA produced during oxidation may be a source of 2-carbon units for elongation of endogenous fatty acids, or elongation to 16: 0 could occur [22] and thus explain the differences found in the tissue 16 :0. De novo synthesis of 16 :0 is known to be inhibited by 18 : 3 (n - 3), both by short-term inhibition of acetyl CoA carboxylase [23] and by a long-term adaptive response in which the activity and synthesis of acetyl CoA carboxylase [24,25] and fatty acid synthetase [24271 are decreased. Furthermore, the malonyl CoA used for elongation of fatty acids during synthesis in the cytosol is probably derived from the same pool providing 2-carbon units for desaturation/elongation of 18 : 3 (n - 3) in the microsome [28]. Microsomal elongation is more active with unsaturated than saturated fatty acid substrates [28]. It is possible, therefore, that com-
TABLE III Bruin total fatty acid composition and results of 2-way ANOVA for significant differences due to formula content of 18:2 (n - 6) and 18:3 (n - 3)
Data represent mean f SE. (n = 6). Where P-value indicates a significant interactive effect of formula 18 : 2 (n - 6) and 18 : 3 (n - 3), individual group comparisons, determined using Fisher’s least significant difference, with Bonferroni correction, are given. % of total fatty acids % 18:2(n-6)
16
% 18:3(n-3)
1
P-value 2 30
4
1
18:2(n-6)
18:3 (n -3)
interaction
4
16:0 18:0 ZSats
21.1 f0.5 22.8iO.3 46.6+0.5
19.6 + 0.2 23.6fO.l 45.7kO.2
19.0f0.4 23.4 i 0.1 45.OkO.3
18.7kO.4 24.2 f 0.2 45.6 + 0.4
0.0006 0.004 0.02
0.03 0.0006 NS
NS 1 NS NS
16:l(n-7) 18:1(n-9) Z Monos
2.0*0.1 20.4iO.2 24.6iO.2
1.7kO.l 19.7 f 0.3 23.7kO.4
1.5 kO.1 19.8+0.3 23.6 f 0.4
1.4iO.l 18.5kO.l 22.2io.2
0.0006 0.001 0.0004
0.05 0.0006 0.001
NS NS NS
18:2(n-6) 20:4(n-6) 2 (n - 6)LCP
1.5 io.0 10.3 f 0.3 16.5 f 0.7
1.6kO.O 10.3 * 0.2 16.2kO.2
1.8+0.1 ‘.a 11.2*0.2 19.0 + 0.2
2.3 + 0.1 b 10.6iO.2 17.0*0.3
< 0.0001 0.007 0.0004
< 0.0001 NS 0.008
0.002 NS 0.05
0.1 *o.o 8.8iO.l 9.3 +0.2
0.2*0.1 10.3 + 0.2 11.0*0.2
tr 8.6kO.2 8.9 & 0.2
0.1+ 0.0 10.3 f 0.3 11.0*0.3
0.05 NS NS
NS < 0.0001 < 0.0001
NS NS NS
25.8*0.6 18.0k0.0 154.3 + 2.4
27.2 f 0.3 18.0 f 0.0 162.7 f 1.4
27.9 k 0.2 18.1+ 0.0 162.7 f 0.7
28.0 f 0.4 18.1 fO.O 166.0 f 1.8
0.002 NS 0.002
NS NS 0.002
NS NS NS
18:3 (n -3) 22:6(n-3)
,z (n - 3)LCP PLCP MChL UI
’ See footnote Table II for abbreviations ’ Values in the same row with different superscript letter are significantly different (P < 0.01).
Fig. I. Plots to show 16:0,
1X: 1, 1X:.! (n -6)
and 22:6
total fatty acids) in: (a) liver total phospholipid: fatty 16/4;
acid of piglets hatched,
30/l;
fed formula
containing:
and black. 35/4
YIX:1
(n -3)
(5
and (b) brain total
crossed. (II -6)/“ilX:3
16/l:
clear, (II -3)
are given in Tables II and III.
these fatty acids in piglets fed the high 18:3 (n - 3) formulas. In fact, liver PL and brain levels of 16: 1 (n - 7) and 18: 1 (n - 9) were lower in piglets fed 4% compared to 1% 18: 3 (n - 3) in formula (Tables II, III). Possibly this could be explained by inhibition of A”-desaturase by 18: 3 (n - 3) [291. Significant changes in the 2 monounsaturated fatty acids, predominantly 18: 1 (n ~ 9), of brain and liver were seen in response to formula 18 : 2 (n - 6). The changes attributed to formula 18: 2 (n - 6), however, are probably most appropriately discussed with respect to formula content of 18: 1 (n - 9). Liver PL and brain TFA 18: 1 (n - 9) were about 50% and 5% lower, respectively, when formula containing high 18 : 2 (n - 6) (low 18: 1 (n - 9)) rather than low 18: 2 (n - 6) (high 18: 1 (n - 9)) was fed (Tables II, III; Fig. 1 a,b). Possibly, the more limited effect of the formula 18 : 1 (n - 9) and 18: 2 (n - 6) content on brain than liver reflects de novo fatty acid synthesis by the oligodendrocytes which are known to synthesize large amounts of myelin lipid, containing particularly high 18: 1 (n - 9) [19,20], during the first 3 weeks after birth in the piglet [32]. Levels of 18 : 2 (n - 6) were increased from about
16 to 22% of total fatty acids in the liver PI.. but f’rom about 1.5 to 1.8-2.2s of fatty acids in the brain ot piglets fed the high compared to low IX : 2 (II 0) formulas. Thus, it may bc worth considering that the more prominent effects of the formula IX : 2 (II 6) Oil liver 18: 1 (n - 9) may also include the known inhibition of A”-desaturase by IX : 2 (II -- 6) [30,31]. As well as 18:2 (II - h), levels of 20:4 01 ~~~ 6) in the liver PL and brain TFA were incrcascd in response to high formula 18: 2 (II ~ 6) (Tables II. III: Fig. 1 a,h). The brain TFA Cs;,1X: 3 (II -~ 6) demonstrated a f’urthcl increase when 4% 18: 3 (n - 3) rather than 1’7 IX : 3 (n - 3) was fed with high It; : 2 (II .- 6). The unsaturation index of Iivcr Pt. and brain TFA showed independent, significant changes due to the formula 18 : 2 (n - 6) and 18 : 3 01 ~~3) content: a higher unsaturation index was assnciatcd with either high IX: 2 (n ~ 6) or high IX: 3 (12 ~ 3) in the formula (l’;tbles II, III). The change in unsaturation index was more pronounced in the liver than brain, and when the formula IX: 3 (n .- 3) rather than 1X: 2 (II 0) was increased (Table 11). The accompanying change in the liver PL mean chain length is explained by the combination of lower _“ saturated fatty acids and higher long-chain polyunsaturated fatty acids. primarily 27 : h (n - .i), when the high 1X :i (II ~~3) formula wax fed. Alterations in membrane protein activities have been associated with changes in the unsaturation index ol’. for example, total phospholipids of liver microsomal membrane [33] and plasma membrane [34]. Whethel the small, but statistically Ggnificant. change\ in liver fatty acid composition and membrane unsaturation seen in this study are of physiological relevance to the newborn is not known. In contrast to the tivcr. the highly significant (I’ < 0.000 1) incrcasc in brain 22 : h (II - 3) when 4 rather than l’i IX:3 (II 3) W;I~ fed. had no effect on the brain total long-chain polyunsaturated fatty acids or mean chain length. This i%prohablq cxplaincd by the well known rcplaccmcnt of 21: 0 (?I - 3) with 22: 4 (II -. 6) and particular’ll, 37 : 5 (or 6) in central nervous system lipids of (II 3) t’atty acid-dcficient animals [ 15- IS]. This study has demonstrated an inverse relationship between formula 1X:3 (II ~~1) and the c; 16: 0 in structural phospholipid of the liver. A similar relationship occurs in the brain when high IX : 3 (II - 3i is fed with 16% 18: 2 (II ~ 0). This is. to our knowlcdgc, the first report of a potential interaction between the dietary intake of 1X: 3 (II - 3) and deposition of 16 : 0 in growing tissues. The possible mechanisms responsible arc speculative and cannot he determined from the results here. Further studies are required to investigate the effect of formula 18: 3 (II - 3) on dc nova saturated fatty acid synthesis. The effect ~1 dictar) medium-chain fatty acids on the synthesis or elongation of fatty acids also requires investigation. In conclu-
267 sion, determination of essential fatty acid requirements, specifically 18 : 3 (n - 31, should consider possible influences on the non-essential fatty acids which also play a role in determining the properties of structural membranes of important organs such as the liver. Acknowledgments
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