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Articles

Dietary Trans Fatty Acids Combined with a Marginal Essential Fatty Acid Status during the Pre- and Postnatal Periods Do Not Affect Growth or Brain Fatty Acids but May Alter Behavioral Development in B6D2F₂ Mice¹

Department of Health Studies and Gerontology, University of Waterloo, Waterloo, Canada N2L 3G1

²To whom correspondence should be addressed. E-mail: wainwrig{at}healthy.uwaterloo.ca.

	ABSTRACT

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The objective of this study was to investigate whether dietary trans fatty acids (TFA) during the pre- and postnatal periods would exacerbate the effects of marginal essential fatty acid (EFA) status on growth, brain long-chain polyunsaturated fatty acids (LC-PUFA) and behavioral development in B6D2F₂ mice. Pregnant B6D2F₁ females were randomly assigned to one of the following three diets: marginal EFA plus 22% trans 18:1 (mEFA + TFA); marginal EFA (mEFA); and control (CON). The total 18:1 content in all diets was similar. The offspring were weaned and maintained on the same diets. Both the mEFA and mEFA + TFA groups had reduced growth and brain weight compared with CON, but did not differ from one another. As expected, the mEFA and mEFA + TFA groups had reduced docosahexaenoic acid [DHA; 22:6(n-3)]) and increased 22:5(n-6) concentrations in brain phosphatidylcholine (PC) and phosphatidylethanolamine (PE) compared with the CON group, but again did not differ from one another. Reversal learning in the T-water maze was significantly slower in the mEFA + TFA groups compared with the mEFA group and both were slower than the CON group. These findings illustrate that TFA combined with a marginal EFA status do not exacerbate the effects of marginal EFA status on growth or brain LC-PUFA. However, long-term effects of dietary TFA during the pre- and postnatal period on behavioral development and neural function should be investigated in future studies.

KEY WORDS: • dietary trans fatty acids • essential fatty acid status • arachidonic acid • docosahexanoic acid • behavioral development

	INTRODUCTION

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Linolenic [LNA,³ 18:3(n-3)] and -linoleic [LA, 18:2(n-6)] acid are important for normal growth and development (1) . These essential fatty acids (EFA) are converted to long-chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid [DHA, 22:6(n-3)] and arachidonic acid [AA, 20:4(n-6)]. AA and DHA are integral structural components of neural membranes, and AA functions as a neuronal second messenger. EFA deficiency in animals has been shown to contribute to aberrations in cognitive development and alterations in LC-PUFA composition of membrane phospholipids in the nervous system (2 ,3) .

Dietary trans fatty acids (TFA) are derived mainly from hydrogenated vegetable oils. During pregnancy and lactation, TFA are transferred to the developing fetus (4 ,5) as well as to human milk through maternal diets (6 ,7) . Some studies have suggested that TFA may have adverse effects on the developing fetus and infant (4 ,8) with respect to growth and development. The putative mechanism for this is based on the observation that TFA may impair the desaturation and elongation of LA and LNA to their LC-PUFA such as DHA and AA (9 ,10) . Animal studies have demonstrated elevated brain 22:5(n-6) concentrations (11 ,12) with reduced DHA concentrations (11) after relatively high intakes of TFA. An increase in 22:5(n-6) has been proposed to be a compensatory mechanism for a reduction in DHA (13) , suggesting that brain LC-PUFA may be influenced by TFA.

The presence of TFA in human milk, the observed inverse relationship of TFA with (n-3) and (n-6) fatty acids in human milk (6 ,7) and the possibility that TFA may have an adverse effect on EFA status could have important consequences for preterm infants. Preterm infants are denied the period of peak accumulation of LC-PUFA that occurs in the brain during the last trimester of pregnancy (1) . Several studies have demonstrated that preterm infants benefit from additional dietary LC-PUFA with regard to visual development (14 ,15) . Findings from such clinical trials suggest that preterm infants may experience suboptimal EFA status.

Breast-feeding is becoming increasingly popular in preterm infants and the concentration of TFA in human milk can be as high as 17% (6) . On the basis of studies in rats, it has been suggested that TFA exacerbate the effects of suboptimal EFA status (16) . It is therefore important to investigate whether TFA intensify the effects of a suboptimal EFA status on brain fatty acid composition and behavioral development. Furthermore, no study to date has investigated whether TFA affect neural function. Thus, the objective of this study was to investigate the effects of TFA combined with a marginal EFA status on growth, brain LC-PUFA composition and behavioral development in B6D2F₂ mice.

	MATERIALS AND METHODS

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Animals.

B6D2F₁ parental mice were obtained from Harlan Sprague Dawley, Indianapolis, IN. This F₁ hybrid cross has been used previously in our laboratory in studies that showed retardation of brain and behavioral development due to EFA imbalance (17) . Mice were housed together in groups of 3–5 animals of like sex, with free access to nonpurified diet (Purina Rodent Chow No. 5001 (St. Louis, MO)) and tap water until mating commenced when they were 4–5 mo old. They were maintained at 22 ± 1°C under a reversed 12-h light:dark cycle in standard opaque plastic mouse cages (28 x 18 x 13 cm³) with Beta-Chip hardwood bedding and several sheets of toilet tissue for nesting material. The sample size was 15–16 litters per dietary group for growth and behavioral testing, and 11 mice per group for brain phospholipid analysis.

Experimental design and diets.

Mice were mated between 0900 and 1500 h. Any female with a vaginal plug was subsequently weighed and assigned randomly to one of three study diets varying in EFA and trans fatty acid content. At birth, the litters were culled to six pups; an attempt was made to ensure an equal number of males and females. The ratio of males to females was similar among the three diet groups. The mice were weaned when they were 3 wk old (42 d postconception), and fed the experimental diets until the end of the study when they were 7 wk old. Litters and dams were weighed weekly. The diet groups were as follows: 22% trans fatty acid with marginal EFA (mEFA) diet (calculated composition: 22% 18:1trans, 13% 18:1cis, 2.5% LA, 0.1% LNA) (mEFA + TFA), a marginal EFA diet (calculated composition: 35% 18:1cis, 2.5% LA, 0.1% LNA) (mEFA) and a control diet (calculated composition: 35% 18:1cis, 10% LA, 1% LNA) (CON). The trans fats were added in the form of 18:1 because 18:1 trans isomers represent 90% of dietary trans fatty acid (18) . To prevent confounding on the outcome variables by the high total 18:1 content in the mEFA + TFA group, the total 18:1 content in all diets was maintained at the same level. The marginal EFA diet was designed to contain only sufficient LA to prevent growth retardation, with trace levels of LNA. The fatty acid composition of the diets was modified by mixing oils from various sources in specific proportions based on prior analysis; the source oils were corn, coconut, soybean, hi-oleic sunflower (Dyets, Bethlehem, PA), partially hydrogenated soybean and fully hydrogenated coconut oil (Canamara Foods, Toronto, Canada). The fatty acid composition of these dietary oil mixtures is shown in Table 1 . (It can be seen that the LA content in the mEFA + TFA was somewhat higher than the calculated value, although the values for the other fatty acids were within the anticipated range.) These oil mixtures were then incorporated in appropriate amounts into AIN-93M semipurified fat-free diet from conception to weaning, or AIN-93G semipurified fat-free diet postweaning (19) (Dyets). Mice consumed food ad libitum and food intake did not differ among the diet groups. The diets were made on a weekly basis and stored at 4°C. The diets were coded so that the experimenters were not aware of the diets the mice were receiving. At weaning one male from each litter was kept for future behavioral testing, and one male and one female mouse were anesthetized under Halothane (MCT Pharmaceutical, Cambridge, Canada) and, when completely unconscious, decapitated. The whole brain was removed and weighed. At the end of the study at 7 wk, the male mice were killed. The whole brain was removed, weighed and stored at -80°C until further analysis. The brains excluded the olfactory bulbs anteriorly and 2 mm below the medulla posteriorly. All procedures were approved by the Animal Care Committee at the University of Waterloo, in compliance with the Animals for Research Act of Ontario and the Guide for the Care and Use of Experimental Animals from the Canadian Council of Animal Care.

The behavioral development of one male and one female mouse from each litter was assessed on d 12–13 (d 32 postconception for all mice), using a standardized test battery validated for use in mice [discussed in detail by Wainwright (20) ]. The timing of testing according to postconceptual age rather than birth date is appropriate because the schedule of brain development is not related to the day of birth, which can vary among litters.

Briefly, each pup was tested on 12 tests, measuring such developmental landmarks as righting, cliff aversion, grasping and climbing, eye-opening, visual placing and auditory startle. The mouse was scored on a scale between 0.0 and 1.0 on each test, and a mean overall score was determined on the 12 tests. In addition, a separate average score was determined for those tests with a significant sensory component, e.g., eye-opening, visual placing, auditory startle, as well as for those in which motor skills predominate, e.g., grasping and climbing. At 7 wk of age, one male from each litter was tested on acquisition and reversal learning in a T-water maze, also as described in detail previously (21) . In this task, the mouse was placed in the stem of the T facing toward the base, requiring it to turn around, swim down the stem and choose to swim either left or right to escape to a hidden platform. The position of the platform for the duration of the acquisition learning was determined randomly for each mouse. The criterion for a wrong turn (or error) was that the body, to the base of the tail, had rounded the corner in a direction away from the platform. The mice were allowed to remain in the maze until they found the platform, to a maximum of 60 s. A mouse was considered to have learned the task when it made no errors in a block of five trials. For the reversal learning phase, this protocol was repeated, except that the mice were required to escape on the side opposite to that on which they had learned.

Lipid analysis.

The lipids in whole brains were extracted in chloroform/methanol (1:1) with the use of a method modified from Bligh and Dyer (22) . These lipid extracts were then separated into different phospholipid fractions by TLC on silica gel plates, using a solvent system of chloroform/methanol/acetic acid/water (100:75:7:4). The fatty acids in the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were then esterified with 0.64 mol/L methanolic sulfuric acid and analyzed on a gas chromatograph (Shimadzu GC-17A Gas Chromatograph, Shimazu, Kyoto, Japan), equipped with a flame ionization detector and a 15 m x 0.32 mm x 0.25 µm fused silica capillary column (Supelcowax 10, Supelco Park, Bellefonte, PA). The carrying gas (helium) flow rate was 2.0 mL/min, with a split ratio of 50. The column temperature was held at 150°C for 2 min, then programmed to 210°C at 3°C/min and held for another 10 min. The injector and detector temperatures were maintained at 250°C. Fatty acids were identified via comparison retention times with authentic standard mixtures (Nu-Chek-Prep, Elysian, MN). Data on 11 brains per group are reported for brain fatty acid composition. The reason for this is that two types of silica gel plates were used for the TLC, and successful separations of the PE and PC fractions were not obtained with one type of plate. Thus, values for a number of mice obtained on these plates (n = 5 for mEFA + TFA, n = 4 for mEFA and n = 4 for CON) were eliminated from the analysis.

Statistical analysis.

When there was more than one observation per litter, the data were analyzed using the litter as the unit of analysis. The growth and fatty acid data were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons to compare groups. The behavioral data were not normally distributed and were therefore analyzed by a "robust" ANOVA procedure in which the scores were transformed to ranks (23) . Subsequent preplanned comparisons were performed between mEFA and mEFA + TFA to determine the effects of TFA and between mEFA and CON to determine the effects of a marginal EFA status using Student’s t test, also on the ranked scores. The statistical tests were performed by SYSTAT (version 5, SYSTAT, Evanston, IL). The level of significant difference was P < 0.05.

	RESULTS

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Growth and development.

Litter size and litter weight were not significantly different before weaning among diet groups. At weaning, body weight was significantly greater in CON mice compared with mEFA mice, and mEFA + TFA mice had intermediate weights that did not differ from either of the other groups. Brain weight was greater in the CON group compared with the mEFA + TFA group, and here the mEFA group was intermediate (Table 2 ). Body weight at the end of the study was significantly greater in CON compared with mEFA mice, but not mEFA + TFA mice. At this time, however, brain weight was not significantly different among groups. Brain weight, expressed a proportion of body weight, did not differ among groups at weaning or at the end of the study (data not shown).

Effects of the diets on brain fatty acid composition were observed in both the PE and PC fractions. As expected, mEFA + TFA and mEFA groups had reduced DHA in brain PE and PC and increased 22:5(n-6) in brain PC and PE compared with CON mice, but mEFA and mEFA + TFA groups did not differ significantly from one another (Table 3 and 4 ). There were no significant effects on AA or 22:4(n-6).

No significant differences were found among diet groups in behavioral development determined at d 12–13. Scores were as follows: CON (n = 16 litters), 0.65 ± 0.01; mEFA (n = 15 litters), 0.65 ± 0.01; and mEFA + TFA (n = 15 litters), 0.64 ± 0.01. Separate analyses of the average of the tests that represent "sensory" categories and "motor" categories are shown in Figure 1 . The mEFA + TFA mice tended to have the lowest scores for sensory development, and CON the highest, with mEFA intermediate (P = 0.10; one-tailed).

View larger version (30K): [in this window] [in a new window]   Figure 1. Scores on motor and sensory development at d 12–13 in B6D2F2 mice fed marginal essential fatty acid plus trans fatty acid diet (mEFA + TFA), marginal essential fatty acid diet (mEFA) or control diet (CON) since conception. Values are means ± SEM, n = 15–16 litters (1 male and 1 female per litter).

The number of trials in which the mice made errors before they reached the criterion response (5/5 trials correct) during acquisition and reversal learning are shown in Figure 2 . The mEFA + TFA group had improved acquisition learning compared with mEFA mice but the opposite effect was observed for reversal learning. During reversal learning, the mEFA + TFA group had a significant increase in the number of trials with error compared with the mEFA group. Similarly, mEFA mice had significantly more trials with error compared with CON mice.

View larger version (28K): [in this window] [in a new window]   Figure 2. Number of trials with error during acquisition and reversal learning in a T-water maze in 7-wk-old B6D2F2 mice fed marginal essential fatty acid plus trans fatty acid diet (mEFA + TFA), marginal essential fatty acid diet (mEFA) or control diet (CON) since conception. Values are means ± SEM, n = 15–16. *Significantly different from mEFA; significantly different from CON, P < 0.05. Note that statistical analyses were performed on the scores transformed to ranks.

	DISCUSSION

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Several studies have investigated the effects of TFA on brain fatty acid composition (11 ,12 ,27) . These studies in newborn and suckled piglets and in rats demonstrated elevated 22:5(n-6) (11 ,12 ,27) and reduced DHA (11) concentrations in brain PE as a result of relatively high trans fatty acid intakes (28–50% of total fatty acids), suggesting that brain LC-PUFA may be influenced by TFA. Hill et al. (16) investigated the effects of TFA combined with an EFA-deficient diet. In that study, it was demonstrated that TFA (56% of total fatty acids) intensified EFA deficiency as determined by dermal scores and heart phospholipids in rats. Specifically, the 20:3(n-9)/20:4(n-6) ratio, an indicator of EFA deficiency, was greater in rats fed TFA and an EFA-deficient diet compared with rats fed an EFA-deficient diet alone. Pettersen and Opstvedt (11 ,27) also demonstrated an interaction between dietary TFA and dietary LA and LNA on brain DHA and AA. These studies suggest that the effects of TFA are dependent on EFA status. However, our results did not support an effect of TFA on brain LC-PUFA in mice fed a marginal EFA diet.

There are some difficulties associated with interpretation of the previous studies in that the diets varied in their EFA as well as trans fatty acid contents. Specifically, in these studies, LA, LNA and 18:1 were not balanced among the diet groups. A particular concern with the addition of TFA as 18:1trans is the increase in the total 18:1 content in the diet. 18-Carbon unsaturated fatty acids, including the TFA, inhibit 6 desaturases (28 29 30 31) . Thus, the high 18:1 content only in diets containing TFA may have been a confounding variable in previous studies. Further support for this argument is provided by the DHA data in the present study. In this study, 18:1 levels were high in all groups, and comparison of the PE data with previous work in our laboratory in which dietary 18:1 levels were lower, indicates that the relative levels of DHA in PE are lower than those seen previously (17) . The possibility of such a relationship between dietary 18:1 and tissue DHA becomes then an important question for future study.

As expected, in this study, the mice receiving the marginal EFA diet had lower DHA and higher 22:5(n-6) concentrations in brain phospholipids. Because LA was provided in marginal amounts, no effect on AA in brain PE and PC was observed. In comparison to the mice fed a marginal EFA diet, those fed a marginal EFA diet combined with TFA did not have higher 22:5(n-6) or lower DHA or AA in brain PE or PC. Thus, TFA did not appear to play a role in intensifying the effects of a marginal EFA status on brain LC-PUFA. Our findings are comparable to previous findings, which demonstrated that a level of 2% of LA was sufficient to prevent a reduction of AA by TFA in mitochondrial phospholipids from adult rats fed TFA diets (with balanced total 18:1 content) with varying amounts of LA (24) . One unexpected factor in our study was that the mEFA + TFA diet contained more LA (3.7%) than the mEFA diet (2.6%). However, these levels can still be considered marginal relative to the 9.2% in the control diet. Thus, although it is possible that the effects of TFA may have been reduced somewhat due to the slightly higher LA, this small difference did not result in significant differences in brain LC-PUFA, and it is therefore unlikely that it would have affected the other outcome variables.

Competition of TFA with EFA metabolism has been observed in several studies, but the interactions are complex, and they vary depending on position of the trans bond (32) . Inhibition by TFA was shown to be greater for (n-6) than (n-3) fatty acids (33) . In our study, none of these pathways appeared to be affected by the TFA. One explanation for this could be that desaturase activity is upregulated by the high need for (n-6) and (n-3) LC-PUFA in developing and growing animals, particularly if LA and LNA supplies are marginal, and/or in response to inhibition by TFA. The latter is supported by findings of increased 5 desaturase activity in liver microsomes of rats fed TFA (16) . However, we did not measure desaturase activity in our mice.

Interestingly, although there were no significant differences in brain LC-PUFA composition in the two marginal EFA groups, some trends for differences in behavioral development were observed. Although not significant (P = 0.10; one-tailed), sensory development was lowest in the mice fed the diet containing TFA. Sensory development includes eye opening and visual placing, and studies have suggested that DHA during development is necessary for normal visual development (34) . Interestingly, there was a similar trend for DHA concentrations being lower in mice fed a marginal EFA diet with TFA in both brain PE (P = 0.086; one-tailed) and PC (P = 0.053; one-tailed). Similar to these data, behavioral development, as assessed in the T-water maze, was altered significantly by exposure to dietary TFA. Both groups of mice fed marginal EFA with or without TFA made more errors compared with controls during reversal learning, and, in addition, the mice fed the marginal EFA diet with TFA made more errors during reversal learning than those fed the marginal EFA diet alone. Similar effects on this task have also been seen in mice exposed to prenatal ethanol, accompanied by changes in brain fatty acid composition (21) . The faster acquisition learning in the mice fed marginal EFA with TFA could be the result of decreased strength of lateralization as seen previously in rats exposed to ethanol prenatally (35) . This means that if animals were being trained against their preference (as would be expected to occur for half of the animals with random choice of sides), the acquisition task may be easier for the animals with the weaker preference. In this study, however, brain fatty acid composition was not significantly different between the two marginal EFA groups. Nevertheless, an important point to consider is that, as selective systems in the brain subserve various learning and memory functions, fatty acid changes in specific areas, e.g., hippocampus, might correlate better with behavioral changes than measures based on the whole brain, as was done in this study.

Although TFA have not been found in the brain of newborn or suckled piglets (11 ,27) , low concentrations have been seen in rat brain lipids (36 ,37) . However, the amounts of TFA in the brain are much lower than those present in the diet. Despite this, it is not known whether any incorporation of TFA into the brain might have influenced brain function by altering physical properties of brain lipids that were not related to total DHA or AA content in brain PE and PC.

This is the first study to have investigated the effects of TFA on neural function in an animal model relevant to infant nutrition. The trend in the data on sensory development, together with findings on reversal learning, supports the possibility of an alteration in neural function with pre- and postnatal exposure to TFA. These findings suggest that further studies are necessary to investigate the effects of TFA on neural function in an infant-animal model. They also underscore the importance of including functional measurements such as these with those of brain chemistry in studies of this type.

In summary, our findings illustrate that, in a dietary model in which total 18:1 is balanced across groups, TFA, combined with a marginal EFA status, do not exacerbate the effects on growth or brain LC-PUFA. Neural function may be affected, however, suggesting that future studies of the long-term effects of dietary TFA during the pre- and postnatal period should include measures of behavioral development and neural function with those of brain chemistry.

	ACKNOWLEDGMENTS

 
The authors thank Alicia Wind and Mary Fraser for their assistance with animal care and behavioral testing.

	FOOTNOTES

 
¹ Funded by a grant from the Natural Sciences and Engineering Research Council of Canada to P.E.W; I.P.W. is supported by a Post-Doctoral Fellowship Award from the National Institute of Nutrition, Canada.

³ Abbreviations used: AA, arachidonic acid; CON, control; DHA, docosahexaenoic acid; EFA, essential fatty acids; LA, linoleic acid; LC-PUFA, long-chain polyunsaturated fatty acids; LNA, linolenic acid; mEFA, marginal EFA; PC, phosphatidylcholine, PE, phosphatidylethanolamine; TFA, trans fatty acids.

Manuscript received November 28, 2000. Initial review completed January 24, 2001. Revision accepted February 16, 2001.

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