Chapter 7
Omega-3 Fatty Acids Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 09/23/18. For personal use only.
The Effect of Alpha-Linolenic Acid on Retinal Function in Mammals AndrewJ.Sinclair and Lavinia Abedin Department of Food Science, Royal Melbourne Institute of Technology University, Melbourne, Victoria 3000, Australia
The retinal function of several different species is compromised by a dietary deficiency of α-linolenic acid. A review of the literature revealed that diets containing less than 0.1 g/100g diet as α-linolenic acid could not sustain retinal docosahexaenoic acid levels over a prolonged period and that such diets were associated with a reduced response of the retina to light. In these studies the median α-linoleinic acid intake of control animals was 1.25g/100 g diet. A study on the comparative ability of dietary α-linolenic acid and dietary docosahexaenoic acid to provide for retinal docosahexaenoic acid in the guinea pig found that α-linolenic acid was only approximately 10% as effective as docosahexaenoic acid in this regard.
It has been known for many years that the mammalian brain is rich in lipid and that the gray-matter contains a high proportion of polyunsaturated fatty acids (PUFA) (7), especially arachidonic acid(20:4n-6), docosatetraenoic acid (22:4n-6) and docosahexaenoic acid (22:6n-3). These PUFA are found in levels of about 12, 6 and 22% of gray-matter phospholipid fatty acids, respectively, for most mammalian species (2-4). Subsequent analyses of retinal fatty acids, from a smaller number of mammals, showed that retinal phospholipids were also rich in PUFA, with 22:6n-3 being the predominant fatty acid (5). In contrast to the brain and retina, there is a diverse pattern of PUFA in the phospholipids of other tissues, such as liver and muscle, probably reflecting the wide differences in dietary intake of essential fatty acids between species (4). In other words, despite a wide variation in the dietary intakes of linoleic acid(18:2n-6) and α-linolenic acid (ALA or 18:3n-3) between different species in their natural or normal environment, there
© 2001 American Chemical Society
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80 are processing mechanisms (specific carrier proteins, acyl-transferases and22:6n-3binding proteins) which regulate the composition of brain PUFA. Subsequent studies in experimental animals showed that alteration of brain and retinal fatty acid patterns can be achieved by manipulation of dietary essential fatty acids intake (6). Diets rich in linoleic acid and poor in A L A from oils such as safflower oil can lead to loss of 22:6n-3 from various tissues including the brain and retina. Other workers have shown that if this manipulation is initiated during pregnancy it is possible to achieve a more substantial deficiency of 22:6n-3, since major accretions of PUFA in the brain occur in utero and in the immediate post-natal period in most species (7). In order to effect maximum change, it has sometimes been necessary to conduct the experiment over several generations (8). Although the essential fatty acids were discovered in 1930, the essential physiological role of A L A was not convincingly demonstrated until the early 1970s. In terms of retinal function, Benolken et al. (9) showed that the rod responses of the rat become abnormal with electroretinographic (ERG) a- and bwave amplitudes being dependent on n-3 PUFA supply. Moreover, it was concluded that the specific deficit involved abberant phototransduction due to changes in photoreceptor membranes of the rod outer segment (9,10). In a subsequent publication, these workers reported a study where rats were fed chow for 14 weeks (ie. adult rats) and then fed test diets for 6 weeks. They found that compared with a fat-free test diet, the diet containing 2% ethyl linoleate gave a 30% increase in ERG amplitude and 2% ethyl linolenate gave an 80% increase in ERG amplitude. These data could be interpreted that both n-6 and n-3 PUFA are biologically active in relation to this physiological parameter (10). In the rat and the monkey, diets rich in linoleic acid and deprived of n-3 PUFA result in substantial alterations in the retinal and brain PUFA patterns by the first generation, with significant decreases in 22:6n-3 to approximately 40% of normal levels (6,11). Ward et al. (7) recently achieved greater depletion of 22:6n3 in rats using a system of artificial rearing. In theirfirstgeneration animals, brain 22:6n-3 levels were 50% of control values by 8 weeks of age whereas in the second generation, the 22: 6n-3 values were 1 0% of controls by 8 weeks of age. Leat et al. (8) reported that it was possible to almost completely deplete guinea pig retinal lipids of 22:6n-3 to a value of 0.5% of retinal fatty acids by dietary manipulation over three generations. Concomitant with the 22:6n-3 depletion, there is a compensatory increase in the 22 carbon n-6 PUFA, docosapentaenoic acid (22:5n-6) (6). Interestingly, on such diets the 22:5n-6 accumulates in amounts approximately equal to the losses of 22:6n-3 such that the total 22 carbon PUFA content of the tissues is constant. In the face of the high degree of structural similarity between 22:6n-3 and 22:5n-6, it is surprising to find that substitution of 22:5n-6 for 22:6n-3 is associated with functional changes in the retina and cortex. Replacement of 22:6n-3 by 22:5n-6, as a result of dietary manipulation, is associated with a significant reduction in the activity of Na /K dependent AT Phase activity in the nerve terminals of rat brain +
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81 (11\ alterations in ERG responses (6,9,11), learning and behavioral abnormalities in rats, mice and monkeys (12-15) and reports of ultrastructural changes in the rat hippocampus (16). Since the first reports of Benolken et al. (9), other workers have studied the effects of dietary A L A deficiency on retinal function. Neuringer et al. (17) investigated in greater detail the relationship between n-3 intake and retinal function in rhesus monkeys. In contrast to the early work of Benolken et al. (9) in the rat, they reported that a-wave amplitudes were reduced for both cone and rod responses at 3-4 months of age. However, by 2 years, these differences were no longer present suggesting that the animal may have overcome the effects of the dietary deficiency. A similar time-dependent recovery has also been reported in rats (11), however in the guinea pig the ERG differences due to n-3 deficiency do not recover with time (18). While the ERG signal amplitudes normalized in the older but deficient monkeys, both cone and rod ERGs showed delayed b-wave implicit times (b-wave peak time) suggesting an abnormality in the kinetic aspects of visual processing. More importantly, the finding implies abnormality of post-receptoral or higher order neural processing since the b-wave is considered to reflect post-receptoral activity (19). The effect that 22:6n-3 deprivation had on recovery from light exposure was also measured using repetitive flashes by Neuringer et al. (17). Unlike the effects of n-3 deficiency on ERG amplitudes, which were found to decrease with age, recovery from repetitive flashes was most affected for the bwave and was found to increase with age. The data leads to only one compelling conclusion that n-3 PUFA, and especially 22:6n-3, is essential for some retinal functions. It also suggests that the locus of the lesion involves more than rod outer segment membranes as first proposed (9, 10). In this chapter we summarize the previous studies on ALA-deficiency and ERG function in animals and consider the effect of two strategies aimed at increasing retinal 22:6n-3 levels in guinea pigs. First we used an increasing dose of A L A relative to a constant linoleic acid intake, and second we used two levels of dietary 22:6n-3 supplements provided in conjunction with dietary arachidonic acid.
Materials and Methods Animals and Diets Seventy pigmented female guinea pigs (English Shorthair) were randomly divided into five groups of four-teen animals at 3-weeks of age. The guinea pigs were fed one of five different semi-synthetic diets ad libitum for 12-weeks and were supplemented withfreshcarrots and drinking water containing ascorbic acid (400 mg/L) as described by Weisinger et al. (20). The diets contained 10% (w/w)
82 lipid, supplied by mixed vegetable oils, and each diet was designed to provide 17 % linoleic acid (as % of total fatty acids). In all diets, the lipids were based on mixed vegetable oils. In diet SAF, the main lipid was provided from safflower oil and the linoleic acid: A L A ratio was 323:1 (ALA 0.05 % total fatty acids). In diet CAN, the main lipid was provided from canola oil with a linoleic acid: A L A ratio of 2.3:1 (ALA 7 % total fatty acids). Diet BASE was based on mixed vegetable oils, with a linoleic acid:ALA ratio of 17.5:1 (ALA I % total fatty acids). Diets BASE+LCPI and BASE+LCP3 were similar to diet BASE but they were designed to contain supplementary levels of arachidonic acid (1%) and 22:6n-3 (0.7 %) (mimicking the levels in human breast-milk) or 3 % arachidonic acid plus 2.1 % 22:6n-3, respectively. Arachidonic acid was obtained from ARASCO oil (Martek Bioscience, Columbia, MD), processed from a common soil fungus widely distributed in nature and 22:6n-3was obtained from DHASCO oil (Martek Bioscience, Columbia, MD), processed from a microalgal organism. Fatty acid assays of the five diets showed that the linoleic acid levels achieved the desired range of between 16.1 and 17.2 %. Assays of diet BASE+LCPI, gave arachidonic acid and 22:6n-3 values of 0.87 and 0.59 %, respectively, whereas diet BASE+LCP3 gave 2.74 and 1.80 % of arachidonic acid and 22:6n-3, respectively. At the end of the 12-weeks of feeding, animals were sacrificed by C 0 asphyxiation. Retinae from each eye were removed and washed in ice-cold phosphate buffered saline and stored in 10 mL of chloroform/methanol (2:l,v/v) containing butylated hydroxytoluene (10 mg/L) as an anti-oxidant. 2
Lipid Analyses Following lipid extraction from retina, the total phospholipids were separated from the neutral lipids by thin layer-chromatography (20). The methyl esters of the phospholipid fatty acids were separated by capillary gas liquid chromatography using a 50 m χ 0.32 mm (I.D.) fused silica bonded phase column (BPX70, SGE, Melbourne, Australia). Statistical Analyses Significant differences between dietary groups were tested using a one-way ANOVA for each type of fatty acid. Post-hoc comparisons were made using the Tukey test with a significance level of 0.05.
Results Previous studies on n-3 deficiency and ERG function in the rat, monkey and guinea pigs are listed in Table I. The experiments used fat-free diets, safflower or sunflower oil to induce a state of n-3 deficiency. The control diets used ALA-rich
safflower + canola+ fish oil versus safflower safflower versus canola oil
2 generations, test 3 generation
as above
16 weeks from weaning
Guinea pig, albino n=12/group
as above
Guinea pig, albino n=6/group
20
20
21
safflower versus canola oil
safflower versus perilla oil
19 weeks of age
6-9 weeks of age
6-9 weeks of age
13&28 weeks of age
N O T E : Units for A L A are grams per 100g diet. S O U R C E : Data from references 6, 10, 11, 20-22.
rd
nd
1 generation, test 2 generation
Rat, WKY n=12/group
6 weeks of age
22
sunflower versus soybean oil
2 generations, test 3 generation
Rat, wistar, n=12/group
11
rd
safflower, versus soybean 4-12 weeks oil of age
diet in pregnancy, test offspring
Monkey, rhesus, n=7/group
6
6 weeks
Length of deficiency or age tested
Fat-free versus fatfree+ALA
Diet lipids
14 weeks chow, 40 days diet
Paradigm
Rat, albino, n=12/group
Species
10
Study reference
0.82
1.25
0.82
3.2
0.13
0.0385
2
Control ALA %
0.03
0.1
0.1
0.0025
0.06
0.015
0
Deficien t ALA %
Table I. Animal studies demonstrating effects of n-3 PUFA on retinal function
42
8
12
55
39
13
ND
Retinal DHA, % of control
62
68
48
89
79
79
63
ERG, %of control
84 vegetable oils such as soy, canola or perilla oil and in one case pure A L A was used. The diets were introduced during pregnancy and extended across several generations or commenced at weaning or even were initiated in adult animals. The control A L A intakes ranged from 0.13 to 3.2g/100g diet and the ALA-deficient diets containedfrom0 to 0.1 g ALA/100g diet. In the deficient diets, the linoleic acid levels ranged from 0 to 7.2 g/100g diet. Only two studies included more than one level of n-3 PUFA. Both Wheeler et al. (10) and Weisinger et al. (20) found a dose response relationship existed with increasing A L A level, while the latter study also found that dietary 22:6n-3 did not lead to improved ERG responses over that found with dietary A L A even though the retinal 22:6n-3 level increased by about 30% compared with the ALA-fed group. These studies showed that reduced retinal 22:6n-3 levels were associated with increased retinal 22:5n-6, almost as a replacement for the lost 22:6n-3. Losses of retinal 22:6n-3 from 40 to 90% of the control value were associated with significantly reduced a-and b-wave ERG amplitudes. The reductions in ERG amplitude varied from 20 to 52%. There was no obvious relationship between the extent of 22:6n-3 loss from the retina and reductions in ERG amplitudes in the various studies, although studies in the same species show losses of up to 30% in the a-wave amplitudes once the retinal 22:6n-3 value decreased below a critical value (21).
Strategies to increase retinal 22:6n-3 levels There was no significant difference in the body weights of animals at the start or finish of the experiment. The mean increase in body weight across all groups was 359 ± 46 g. The proportion of total PUFA in the retinal lipids ranged between 41 and 45% regardless of the diet. However, the fatty acid profiles varied as a function of diet group. The main retinal phospholipid fatty acids were 16:0,18:0, 18: ln-9, arachidonic acid, 22:5n-6 and 22:6n-3 (Table II). There were significant increases in the proportions of 22:5n-3, 22:6n-3 and 24:6n-3 and significant decreases of linoleic acid, arachidonic acid, 22:4n-6,24:4n-6,24:5n-6 and 22:5n-6 as the A L A content of the diet increased (Figure 1). The additions of arachidonic acid and 22:6n-3 supplements (diets BASE+LCPI and BASE+LCP3) were associated with significant decreases in 22:4n-6, 22:5n-6 and 24:4n-6 and significant increases in 22:5n-3 and 22:6n-3. Diet BASE+LCP3 resulted in significantly more 22:6n-3 and less 22:5n-6 than did diet BASE+LCPI or diet BASE. Supplementing diets with 22:6n-3 and arachidonic acid resulted in significant increases in the proportions of the 24 carbon n-3 PUFA and significant decreases in the 24 carbon n-6 PUFA.
a
a
3
a
a b
b
b
0.07±0.0 1.25+0. l l 16.35±1.09 0.65±0.05 0.13±0.0Γ
b
a
0.09±0.07 0.47+0.05 8.70±0.76 0.17±0.03 0
b
3
b
b
a
b
a
a
b c a
c
b
c
0.07+0.0Γ 0.59±0.08 17.60±1.05 0.25±0.03 0.14±0.02
b
b
b
b
a
b
1.10±0.12 0.76+0.03** 9.21±0.32 2.85+0.16 9.27±0.73 2.11±0.13 0.23±0.02
BASE+LCPI n=14
c
cd
0.08+0.0Γ 0.65±0.06 9.64±0.63 0.22±0.02 0.05±0.00
b
a
a
a
ab
3
b
a
1.50+0.18 0.73±0.03 9.06±0.27 3.49+0.14 15.16+0.65° 2.34±0.16 0.24±0.02
BASEn=14
a
1.38±0.16 0.74±0.03 8.67±0.22 2.70+0.10 9.28±0.96 2.02+0.10 0.20±0.01
CANn=14
a
1.37+0.15 0.88±0.24 9.09±0.33 3.50±0.10 17.35±0.67 2.59+0.22 0.31±0.04
SAF n=12
Diet Group
b
c
d
d
0.13±0.01 0.82±0.08 25.45+0.5 l 0.41±0.03 0.35±0.05
c
c
d
c
d
ab
c
0.91±0.13 0.70±0.01* 8.81±0.39 2.14±0.12 3.43±0.56 1.31±0.12 0.16±0.02
BASE+LCP3 n=14
NOTE: Results expressed as % of total phospholipid fatty acids, mean±SD. Different superscript letters indicate significant differences between the diets at P
