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INRA, Unité de Nutrition Lipidique, Dijon, France and * Laboratoire de Biophysique Sensorielle, Facultés de Médecine et de Pharmacie, Université d’Auvergne, Clermont Ferrand, France
2To whom correspondence should be addressed. E-mail: chardign{at}dijon.inra.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-linolenic acid [18:3(n-3)] for 12 mo to study the long-term effects of these fatty acids on the electroretinogram (ERG). The diets contained 18:3(n-3) in its natural form at 2.0 g/100 g total fatty acids (C group), partially isomerized 18:3(n-3) [1.3 g/100 g cis 18:3(n-3) + 0.7 g/100 g trans 18:3(n-3); cT group] and the control level of cis 18:3(n-3) with trans 18:3(n-3) [2.0 g/100 g cis 18:3(n-3) + 0.7 g/100 g trans 18:3(n-3); CT group]. The ERG and the levels of trans-isomers of the polyunsaturated fatty acids (PUFA) of retinal and hepatic phospholipids were determined after 3, 6, 9 and 12 mo of feeding the experimental diets. Dietary trans -linolenic acid altered the fatty acid composition of retinal and hepatic phospholipids by significantly increasing the 
19trans-isomer of docosahexaenoic acid. Moreover, dietary trans-isomers of -linolenic acid significantly decreased the b-wave amplitude of the ERG by 9 mo of feeding. We conclude that long-term intake of small amounts of trans-isomers of -linolenic acid could disturb visual function. However, further studies are required to determine the mechanisms responsible for this phenomenon.
KEY WORDS: • rats • fatty acids • electroretinogram • trans polyunsaturated fatty acids
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-linolenic [18:3(n-3)] acids, have double bonds under the cis configuration. However, heat treatment of vegetable oils such as deodorization or frying induces isomerization of the cis double bond into a trans double bond (1
–3
). As a consequence, trans PUFA are present in different oils (4
–6
) and food products (7
,8
) consumed by humans. Previous animal studies have shown that the trans-isomers of linoleic and -linolenic acid are desaturated and elongated, forming trans-isomers of arachidonic [AA; 20:4(n-6)] (9
,10
), eicosapentaenoic [EPA; 20:5(n-3)] and docosahexaenoic acids [DHA; 22:6(n-3)] (11
,12
). Possible detrimental effects of trans fatty acids include low birth weight (13
), reduction of head circumference in children (14
), promotion of cardiovascular disease in adults (15
,16
) and impairment of the biosynthesis of long-chain PUFA (17
,18
).
The central nervous system contains mainly lipids rich in (n-6) and (n-3) PUFA. Because these lipids are structural and not related to energy metabolism, they contribute directly to the functioning of cerebral membranes. In the retina, a high level of DHA, the most abundant (n-3) fatty acid in neural tissues (19
), is found in the photoreceptor cells, especially in the photoreceptor outer segments. The retina is not protected against long-chain trans fatty acid incorporation (20
). Because dietary modifications of (n-3) fatty acids alter retinal function as measured by the electroretinogram (ERG) (21
), the question of the effects of trans fatty acids on the visual function should be considered.
Only one of our previous studies considered such possible effects (22
). Rats were fed trans -linolenic acid ethyl esters for 6 wk and the ERG b-wave amplitude was measured using an isolated perfused rat retina as a model. These in vitro data showed that dietary trans-isomers of (n-3) fatty acids significantly altered the maximal b-wave amplitude of the electroretinographic response. However, measuring the ERG in vivo would represent a more physiologic approach and we also thought that a longer feeding period should be tested. Here, we report the effects of isomerization of 18:3(n-3) on the ERG recorded in vivo in rats. Because neuronal tissues are relatively insensitive to dietary changes, a 1-y feeding period was used to allow sufficient incorporation of dietary trans fatty acids into the retina.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All animal use and care were conducted according to the French legislation (authorization A21200 and 3273). Rats were housed in animal quarters under controlled temperature (21 ± 1°C) and light conditions (12-h light:dark cycle). Light intensity measured at various locations of the animal quarters was < 20 lx. Three groups of weanling male Wistar rats (Janvier’s breeding, Le Genest-St-Isle, France) consumed ad libitum three standard diets for 12 mo as previously described (23
) with the following modifications: casein, 180 g/kg; cornstarch, 460 g/kg; and sucrose, 230 g/kg. Diets differed only in their cis and trans -linolenic acid isomer concentrations. The control diet contained 18:3(n-3) in its natural form at 2.0 g/100 g total fatty acids (C group); in another diet, part of the 18:3(n-3) was isomerized [1.3 g/100 g cis 18:3(n-3) + 0.7 g/100 g trans 18:3(n-3); cT group]. The third diet contained a control level of cis 18:3(n-3) and trans 18:3(n-3) [2.0 g/100 g cis 18:3(n-3) + 0.7 g/100 g trans 18:3(n-3); CT group]. Diets were prepared from oil mixtures kindly provided by Lesieur (Coudekerque-Branche, France). Briefly, bleached canola oil was used after deodorization for 52.5 h at 205°C under 3 mbar (24
) to provide trans -linolenic acid. To balance the trans linoleic acid levels in the mixtures, isomerized sunflower oil (270°C, 18 h) was added. The detailed composition of the dietary lipids is presented in Table 1
.
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The ERG was recorded in vivo after 3, 6, 9 or 12 mo of treatment. The procedures used were adapted from those described by Doly et al. (25
). Before ERG recording, rats were dark-adapted for at least 3 h. All further procedures were carried out under dim red light (
> 650 nm) at a constant temperature of 25°C. Rats were anesthetized with an intramuscular injection of ketamine (120 mg/kg body) and xylazine (6 mg/kg body) in a saline solution. The left pupil was dilated with 0.5% tropicamide (Ciba Vision Ophthalmics, Blagnac, France). An irrigating solution (BSS, Laboratoires Alcon, Rueil Malmaison, France) was used to prevent corneal desiccation. After 10 min, the corneal electrode was installed. The ERG was recorded via the corneal electrode (thin silver wire with a 3-mm ring end) and a reference placed on the rat’s tongue. The ERG response was amplified using a low-pass filter setting of 1 Hz and a high-pass filter of 1000 Hz. After amplification, the signal was digitized and processed. The retina was stimulated by a photostimulator (model PS33 PLUS, Grass Telefactor, Astro-Med West Warwick, RI) delivering light flashes (white light, 10 µs, 237 lx) to the eye by optical fibers and a white sphere that mimics a Ganzfeld. One flash was delivered every minute and the mean of 10 individual ERG was considered to be one measurement. The b-wave amplitude of the ERG was determined for each recording and was measured from the peak of the a-wave.
Tissue collection and lipid analysis.
One week after the electroretinographic measurement, rats were killed by decapitation. The liver was removed, eyes were enucleated and both retinas from a single rat were pooled. Total lipids from retinas and liver were extracted according to the Folch procedure (26
). Phospholipids of the retina and liver were separated from neutral lipids by the method of Juanéda and Rocquelin (27
). Phospholipids were transesterified with boron trifluoride in methanol (70 g/L) according to Morrisson and Smith (28
). The fatty acid methyl esters (FAME) were analyzed on a Hewlett-Packard (Palo Alto, CA) 5890 series II gas chromatograph equipped with a spitless/split injector, a flame ionization detector, and a BPX 70-silica capillary column (120 m x 0.25 mm i.d. film thickness 0.25 µm; SGE, Melbourne, Australia). The injector and detector were maintained at 250 and 280°C, respectively. Hydrogen was used as a carrier gas (inlet pressure 300 kPa). The oven temperature was fixed at 60°C for 1 min, then increased from 60 to 175°C at a rate of 20°C/min and kept at this temperature until the end of the analysis. FAME were identified by comparison with commercial or synthetic standards and quantified using the DIAMIR software (JMBS, Portage, MI).
Statistical analysis.
Results are expressed as mean ± SEM. Statistical analyses were performed using the Statistical Analysis System (SAS Institute, Cary, NC). Groups were compared at each time point by ANOVA and the Newman-Keul’s test. Within each group, successive times were compared by Student’s t test. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The diets did not affect the level of 22:5(n-6) in the retina (Table 2
). After 3 mo, the levels of 20:4(n-6) were significantly higher in the cT-group compared with the C- and CT-groups. After 6, 9 and 12 mo of feeding, the CT-group had significantly less retinal 20:4(n-6) than the other groups. The levels of 18:3(n-3) and 20:5(n-3) did not differ among groups or change over time. After 3 mo, the level of 22:6(n-3) was significantly higher in the liver of the CT-group than in that of the C- and cT-groups. After 6, 9 and 12 mo, the cT-group had less 22:6(n-3) than the other groups in both liver and retina. After 12 mo, the difference in 22:6(n-3) between C- and cT-groups represented 2.8 g/100g of total fatty acids in the retina.
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15trans and its desaturation and elongation products, the 20:5 17trans and the 22:6 19trans. The 22:6(n-3) trans-isomer levels were higher in the cT-group than in the CT-group at 9 mo of dietary treatment in the retina and at 12 mo in the liver. Electroretinographic study.
Between 3 and 6 mo, the b-wave amplitude decreased significantly in all groups (Table 3
). It did not change between 6 and 9 mo in the C- and cT-groups, but it decreased in the CT-group. As a consequence, the b-wave amplitude was significantly lower in the CT-group than in the C-group and it was intermediate in the cT-group at 9 mo. Between 9 and 12 mo, the b-wave amplitude did not change in the C- and CT-groups, whereas it decreased in the cT-group. Hence, at 12 mo, the amplitude was significantly lower in both groups fed trans fatty acids than in controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-linolenic acid decreased the ERG b-wave amplitude in rats (22
). In the present study, the experimental conditions were more physiologic, i.e., the electroretinographic study was carried out in living animals and trans fatty acids were given as triacylglycerols, representing only 0.7 g/100 g of total dietary fatty acids. This amount was chosen to reflect that currently found in the human diet (29
). A long-term study was used to allow maximum incorporation of trans PUFA into the retina. We focused on the b-wave (Fig. 1
) because its genesis depends on the integrity of the photoreceptors and the synaptic transmission between photoreceptors and other retina cells (30
). Therefore, the b-wave amplitude is the best index of the quality of retinal metabolism.
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-linolenic acid resulted in the incorporation of 19trans-isomer of DHA into retinal phospholipids, confirming earlier findings of our laboratory (20
). Furthermore, the presence of trans-isomers of DHA in the retina was associated with the reduction of the ERG b-wave amplitude compared with the control group, in which no trans DHA was detected. Isomerizing part of the -linolenic acid (cT-group) decreased the cis DHA levels of the liver and retina. This observation can be explained by the 18:2(n-6)/18:3(n-3) ratio, which was greater than in the control diet (9.5 in the C diet and 14.6 in the cT diet). The deficiency of (n-3) fatty acids has already been associated with a reduction of the ERG b-wave amplitude (21
). However, this was associated with an increased level of 22:5(n-6). Because the level of 22:5(n-6) was unaffected in the cT group, the decrease in the b-wave amplitude of the ERG observed in this group may not be a consequence of the unbalanced dietary (n-6)/(n-3) PUFA ratio. This was confirmed by the results in the CT-group in which the (n-6)/(n-3) PUFA ratio was restored to that of the control diet and the ERG results were similar to those in the cT group, although the decrease in the ERG b-wave amplitude occurred sooner. Retinal but not hepatic AA levels were reduced in the CT group at 6, 9 and 12 mo. Because this reduction did not occur in the cT group, the presence of trans PUFA likely was not the only factor involved. The only difference between the cT and CT diets was the level of cis 18:3(n-3). As a consequence, the total 18:3(n-3) level (cis + trans form) was raised to 2.7 g/100 g of total fatty acids, which increased the (n-6)/(n-3) PUFA ratio. The modified (n-6)/(n-3) PUFA ratio might be related to the modifications of AA, but the mechanisms involved are unknown.
In conclusion, we demonstrated in living animals that long-term intake of small amounts of trans -linolenic acid disturbs visual function. The changes in retinal AA in the CT-group suggest that it is important to prevent the consumption of trans-isomers of -linolenic rather than to correct the unbalanced (n-6)/(n-3) PUFA the ratio by increasing the level of the natural isomer. However, further investigations are required to clarify the molecular mechanisms involved.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AA, arachidonic acid; C group, 18:3(n-3) in its natural form at 2.0 g/100 g total fatty acids; cT group, 1.3 g/100 g cis 18:3(n-3) + 0.7 g/100 g trans 18:3(n-3); CT group, control level of cis 18:3(n-3) and trans 18:3(n-3) [2.0 g/100 g cis 18:3(n-3) + 0.7 g/100 g trans 18:3(n-3)]; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; ERG, electroretinogram; FAME, fatty acid methyl esters; PUFA, polyunsaturated fatty acids; 18:3 15trans, 9cis, 12cis, 15trans-18:3; 20:5 17trans, 5cis, 8cis, 11cis, 14cis, 17trans-20:5; 22:6 19trans, 4cis, 7cis, 10cis, 13cis, 16cis, 19trans-22:6.
Manuscript received 19 February 2002. Initial review completed 8 May 2002. Revision accepted 23 July 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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