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Alterations of eating patterns in industrialized countries have resulted in the increased use of processed vegetable fats and oils obtained by processes of partial hydrogenation, which have substantial amounts of trans and saturated fatty acids (1). It is well established that fatty acids containing trans double bonds, when fed to rats, are readily absorbed and incorporated into tissue lipids (2, 3), and these seem to influence the concentrations of LC-PUFA (4, 5) with still unknown physiologic consequences. Some studies on the effects of these fatty acids on delta-6 fatty-acid desaturase activity have indicated an inhibitory effect, but much of the literature (6) gives results obtained with systems deficient in EFA or using amounts of specific trans fatty acids far from the real-life dietary patterns (7).

Despite the fact that fetal life is a particularly vulnerable period, few studies have dealt with the effects of dietary trans fatty acids on pregnancy and fetal growth and development. Although the conversion of EFA into PUFA in pregnancy may be critical for the fetus and preterm babies, no experimental studies have examined the effects of trans fatty acids on tissue delta-6 fatty-acid desaturase activity. Also, studies considering the transfer of trans isomers across the placenta have reported contradictory results; Pettersen and Opstvedt (8) did not find significant percentages of tran s fatty acids in lipids of newborn pigs for which the mothers were fed high trans diets, and Johnston et al. (9, 10) showed similar results in carcass lipids of newborn rats and in tissue lipids of human fetuses and newborns, suggesting that trans fatty acids do not efficiently cross the placenta unit. In addition, the Federal Drug Administration report of 1985, prepared for the Center for Food Safety and Applied Nutrition, concluded that the human placenta acts as a barrier for trans fatty acids, and this report excluded a significant untoward effects for the fetus (11). However, Koletzko and Muller (12) have rejected this thesis because they detected trans fatty acids at similar concentrations in cord blood of term infants and maternal plasma lipids, and Houwelingen and Hornstra (13) observed a direct correlation between trans-9-octadecenoic acid in the plasma of mothers and in fetal tissues.

Because of the increasing interest in the role of LC-PUFA during fetal and early development, and particularly DHA and AA fatty acids (14, 15) and the potential adverse effects of trans fatty acids on LC-PUFA synthesis, the objective of the present study was to evaluate in a controlled fashion the effect of dietary trans fatty acids on the LC-PUFA status in pregnant rats, placentas, and their fetuses, as well as to determine the delta-6 fatty-acid desaturase activity of liver microsomes of pregnant rats after 10 wk of feeding on three experimental diets containing very low (approximately 0%), high (15%), and very high (30%) trans fatty acids but containing the same proportion of linoleic (18:2 n-6) and α-linolenic (18:3 n-3).

METHODS

Study design.

The protocol of this study was approved by the Animal Laboratory Service of the University of Murcia, and animals received humane treatment according to the regulations for Animal Research of the European Union. Female Wistar rats at weaning (21 d of age), supplied by the Animal Laboratory Service of the University of Murcia (Spain), were housed individually in cages in a room with controlled temperature (22°C) and light period (0800–2000 h). The rats, randomly assigned to three groups of six rats per group, matched by weight, were allowed free access to feed and water, and were fed 10 wk on isocaloric (417 kcal/100 g diet), semisynthetic diets containing (g/kg): casein 200, DL-methionine 10, fat mixture 100, saccharose 100, starch 418, cellulose 80, choline bitartrate 2, mineral supplement 50, and vitamin supplement 50. Composition of the minerals and vitamins supplemented complied with the AIN recommendations (16). The fat consisted of: a mixture of 75% olive oil and 25% soybean oil, with a total trans content of approximately 0%, for the control or C group (very low trans diet); 37.5% olive oil, 34% shortening, and 28.5% soy oil, with a total trans content of 14.5% (high trans diet), for the A group; and 68% shortening and 32% soy oil, with a total trans content of 29% (very high trans diet), for the B group. The diet classification was established on the basis of the trans fatty acid content in human diets. The fatty acid composition of the three diets is summarized in Table 1. The diets were prepared and stored at −4°C.

Table 1 Selected fatty acid composition of diets
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Sample preparation.

After 7 wk on their respective diets, female rats were mated 1:1 in their cages. On d 1 of pregnancy, as indicated by the presence of a mating plug, the male rats were removed and the female rats remained in their cages throughout the pregnancy period. On d 20 of pregnancy, the animals from each group were killed. Immediately on removal, the livers of pregnant rats were rinsed in cold saline solution, and microsomes were obtained as described by Philipp and Shapiro (17). The maternal plasma, brain, and placentas and the fetal livers and brains (four fetuses and placentas per rat) were pooled for each experimental group, frozen in liquid nitrogen, and stored at −80°C until analyzed.

Analytical methods.

Total lipids from different tissues were extracted according to the method of Folch et al. (18). FAME were prepared with methanolic: HCl 3N (Supelco, Bellefonte, PA) at 85°C for 1 h and dissolved in hexane. FAME were analyzed by GLC using an SP-2560 fused silica capillary column (100 m × 0.25 mm inner diameter, 20 μm film thickness; Supelco) in a Hewlett-Packard 5890 gas chromatograph. The oven temperature was programmed 39 min at 175°C and was increased at a rate of 3°C/min to 230°C for 14 min. Helium was used as the carrier gas at a pressure of 42 psi. Peaks were identified by comparison of their retention times with appropriate FAME standards purchased from Sigma Chemical Co. (Urbana, IL). Finally, the double-bond position of isomers were confirmed by GLC–mass spectrometry analysis of their 2-alkenyl-4,4-dimethyloxazoline derivatives, prepared by the procedure of Zhang et al. (19).

For GLC–mass spectrometry analysis, we used a VG Analytical MS System (model 7070EQ, VG Analytical, Manchester, England), equipped with an 11/250 data system interfaced to a Varian GLC (model Vista 6000, Varian Associates, Palo Alto, CA) and operated at an ionization energy of 70 eV. The GLC separation of the 2-alkenyl-4,4-dimethyloxazoline derivatives was performed on the same SP-2560 capillary column described for the FAME analysis. Helium was the carrier gas. The GLC column oven temperature was programmed from 140°C at 1.5°C/min to 220°C for 15 min.

For the final determination of the composition of the 18:1 isomers in the diet, the FAME were fractionated by silver-nitrate thin-layer chromatography and further GLC, according to the procedure to Ulberth et al. (20), to avoid the overlap of trans-12-octadecenoic acid, trans-13-octadecenoic acid, and trans-14-octadecenoic acid with 18:1 cis isomer peaks.

The liver microsomes were also assayed for delta-6 fatty-acid desaturase activity, as previously described by Girón et al. (21), using 0.5 mg of protein of microsomal suspension and studying the conversion of 75 nmol of [1-14C]linoleic acid, specific activity 0.58 μCi/μmol, to [1-14C]α-linoleic acid (results expressed as picomole per minute per milligram of protein).

Statistical methods.

Data were expressed as mean ± SEM. Because fatty acid percentages usually do not follow a normal distribution, data were transformed to their decimal logarithms. One-way ANOVA followed by a posteriori test of multiple range of Bonferroni was used for statistical evaluation of significant differences between groups, using the Statgraphics 7.0 (Statistical Graphics, Cambridge, MA) software.

RESULTS

The average number of fetuses per pregnant rat did not differ significantly between dietary groups (0%, 14.5%, and 29%trans fatty acids): C, 12 ± 1; A, 14 ± 1; B, 11 ± 1).

Table 2 shows some selected fatty acid indices for plasma, liver microsomes, and brain of pregnant rats. Total trans concentrations were significantly higher in the A and B groups compared with the C group in both plasma and liver microsomes, and there was a significant correlation between the percentage of those fatty acids in the diet and their relative contents in both tissues (r = 0.92, p < 0.0001;r = 0.96, p < 0.0001, respectively). The trans isomers identified were similar to those present in the diet. The main trans isomer detected was 18:1 trans, which behaved as total trans fatty acids (results not shown). Total SFA, MUFA, and PUFA remained mostly unaffected in plasma, but the SFA proportion was lowered significantly in liver microsomes of A and B groups parallel to the trans fatty acid intake; in contrast, the MUFA proportion was significantly higher in the B group compared with the C group (Table 2).

Table 2 Fatty acid indices in plasma, liver microsomes, and brain of pregnant rats fed diets differing in their trans fatty acid content
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The brain fatty acid composition of pregnant rats remained fairly constant regardless of the trans fatty acid dietary content. Total trans fatty acids did not surpass 0.6%; nevertheless, the total trans fatty acid proportion was significantly higher in rats fed the A and B diets compared with the C group, the concentration of which was nearly 0%. Trans MUFA were also the most representative trans fatty acids in that tissue (Table 2).

The 18:2 n-6 proportions rose significantly in plasma, liver microsomes, and brain of pregnant rats, parallel to the dietary content of trans fatty acids, and 22:6 n-3 was the most affected fatty acid, with significantly lower concentrations in plasma and liver microsomes for the B group compared with the C and A groups, excepting brain (Fig. 1). The 18:3 n-3 concentrations did not change in any of the above-mentioned tissues because of trans dietary intake (results not shown). The relative percentage of 20:4 n-6 remained unaffected in liver microsomes, but showed a trend to being lower in the plasma of animals fed the trans-containing diets. Moreover, 20:4 n-6 was significantly lower in the brain of the B group (Fig. 1). No significant changes were observed for 20:5 n-3 and 22:5 n-3 in those tissues in pregnant rats (20:5 n-3: plasma: C, 0.68 ± 0.17; B, 1.21 ± 0.36; A, 0.48 ± 0.05; liver microsomes: C, 0.53 ± 0.11; B, 0.40 ± 0.05; A, 0.42 ± 0.05; brain: C, 0.20 ± 0.08; B, 0.34 ± 0.07; A, 0.12 ± 0.05; 22:5 n-3: plasma: C, 0.50 ± 0.03; B, 0.52 ± 0.02; A, 0.41 ± 0.04; liver microsomes: C, 0.62 ± 0.02; B, 0.60 ± 0.03; A, 0.56 ± 0.03; brain: C, 0.16 ± 0.00; B, 0.15 ± 0.01; A, 0.16 ± 0.01).

Figure 1
figure 1

Percentages of linoleic acid (18:2 n-6), AA (20:4 n-6), and DHA (22:6 n-3) in plasma (top), liver microsomes (middle), and brain (bottom) of pregnant rats fed control and 14.5% (A) and 29% (B) trans fatty acid diets for 10 wk;n = 6 for each dietary group. *p < 0.05 vs control; †p < 0.05 vs A group.

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Trans fatty acid concentrations rose in placenta and in fetal liver and brain parallel to the trans content in the diet (Table 3). However, the proportions of those fatty acids were higher in placenta, followed by liver and brain; this last organ reached only about 1% t rans fatty acids in group B. Trans MUFA were the most important t rans fatty acids in all organs studied and behaved as total t rans fatty acids. SFA did not change in any tissue excepting a slight decrease observed in placenta for the A group and in brain for the B group compared with the control group. Similarly, MUFA proportions were maintained fairly constant in all groups and tissues. However, total PUFA were higher in the A and B groups than in the C group in placenta but not in fetal liver and brain, in which they were unaffected. LC-PUFA n-6, and particularly 18:2 n-6, were the fatty acids responsible for that rise (Fig. 2). On the contrary, LC-PUFA n-3 was significantly decreased in the B group in placenta and fetal liver, 22:6 n-3 being the main fatty acid affected (Fig. 2), although the proportions remained unchanged in fetal brain. No significant changes were observed for 20:5 n-3 and 22:5 n-3 in placenta and those tissues (20:5 n-3: placenta: C, 0.14 ± 0.01; B, 0.11 ± 0.01; A, 0.12 ± 0.02; fetal liver: C, 0.28 ± 0.03; B, 0.21 ± 0.01; A, 0.33 ± 0.10; fetal brain: C, 0.10 ± 0.03; B, 0.05 ± 0.01; A, 0.10 ± 0.05; 22:5 n-3: placenta: C, 0.44 ± 0.05; B, 1.04 ± 0.63; A, 0.44 ± 0.05; fetal liver: C, 0.23 ± 0.02; B, 0.25 ± 0.03; A, 0.41 ± 0.07; fetal brain: C, 0.12 ± 0.01; B, 0.10 ± 0.00; A, 0.11 ± 0.01).

Table 3 Fatty acid indices in placenta, liver, and brain of fetal rats whose mothers were fed diets differing in trans fatty acid content
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Figure 2
figure 2

Percentages of linoleic acid (18:2 n-6), AA (20:4 n-6), and DHA (22:6 n-3) in placenta (top) and liver (middle) and brain (bottom) of fetal rats whose mothers were fed for 10 wk on 0% (Control), 14.5% (A), and 29% (B) trans fatty acid diets;n = 6 for each dietary group. *p < 0.05 vs control; †p < 0.05 vs A group.

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Figure 3 shows the liver microsomes delta-6 fatty-acid desaturase activities of pregnant rats. The activity was significantly inhibited in A and B groups compared with the control, and no differences were found between A and B groups.

Figure 3
figure 3

Delta-6 fatty-acid desaturase activity (pmol·min−1·mg−1 protein) in liver microsomes of pregnant rats fed for 10 wk on 0% (C), 14.5% (A), and 29% (B) trans fatty acid diets;n = 6 for each dietary group. *p < 0.05 vs C.

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DISCUSSION

The consequences of an exposure of the fetus to trans fatty acids or their effects on the maternal-fetal transfer of EFA and LC-PUFA, which occur during pregnancy, are not clear. In the current study, no changes were found in the number of fetuses per pregnant rat. Buison et al. (22) did not find any reduction in the litter size and weight in pregnant rats fed soybean oil and vegetable shortening with respect to those fed palm oil. However, Hanis et al. (23) reported a reduced litter size in the group of rats receiving trans fatty acids, but they used very low concentrations of 18:2 n-6 in their trans diets (0.38% and 0.54% of total energy of diet), which limits their conclusions and raises questions as to the safety of high intake of dietary trans isomers during pregnancy.

Although placental transfer of saturated and cis unsaturated fatty acids have been demonstrated in rats, very few studies have investigated the placental transport of trans fatty acids, and the results obtained are contradictory (8, 9, 11, 12). The current study shows that trans fatty acids are incorporated into plasma and liver microsomes of pregnant rats in high concentrations and according to their profile and content in the diets; however, this is shown not to occur in brain, reflecting a clear protective mechanism to limit the incorporation of these fatty acids in the CNS. The placenta also incorporated high amounts of trans isomers into its structure. However, this barrier was not completely impermeable, inasmuch as a number of trans fatty acids crossed the organ and accumulated in the liver of the fetus, showing a clear exposure of fetal tissues to maternal dietary trans fatty acids.

The amount of fetal brain incorporation of trans fatty acids proved to be small (1.18% for the B group fed high trans diet), but this amount was slightly higher than the concentration found in the pregnant mother's brain, suggesting a low transference of trans isomers to the CNS during early development and possibly a further metabolism as previously reported by Moore and Dhopeshwarkar (24). These authors injected a 14C-labeled albumin complex of elaidic or linoelaidic acid into the jugular vein of pregnant rats and showed 0.06% radioactivity incorporation for both fatty acids in the fetal brain. They also reported the formation of radioactive palmitic acid in rat fetal tissue from rats injected with labeled elaidic acid, suggesting this last fatty acid is β-oxidized. Pettersen and Opstvedt (8) found concentrations of < 0.1% of trans fatty acids in brain phosphatidyl-ethanolamine of newborn piglets, a fact that supports the idea that low concentrations of trans isomers might accumulate in the brain structure.

The pregnant mother is a very important source of DHA for the fetus (25), and the placenta reaches high concentrations of DHA in its triglycerides (26), part of which is transferred to the fetus. In the current study, the incorporation of trans isomers in the tissues of pregnant and fetus rats increased 18:2 n-6 and decreased DHA tissue concentrations. Currently, it is known that DHA may be formed from 22:5 n-3 by a metabolic pathway mediated by delta-6 fatty-acid desaturase activity and retroconversion of 24:6 n-3 to 22:6 n-3 (27). Although AA and LC-PUFA n-6 did not change significantly in those tissues, they showed a tendency to decrease, which agrees with the results of partial inhibition obtained on in vitro delta-6 fatty-acid desaturase activity of liver microsomes of pregnant rats fed the experimental diets containing appropriate amounts of EFA.

Blomstrand et al. (28) did not find any variation in delta-6 fatty-acid desaturase activity in rats fed partially hydrogenated vegetable and marine oils, supplemented with linoleic acid, but their rats were normal adults rather than pregnant rats. During pregnancy, animals have an intense metabolic activity to supply all necessary nutrients to the fetus, particularly EFA and LC-PUFA. Although the supply of linoleic acid was sufficient in the three diets, the trans fatty acids might compete with the EFA, resulting in an inhibition of delta-6 fatty-acid desaturase activity with the subsequent tissue accumulation of these substrates and decreased concentrations of their long-chain derivatives. We cannot exclude that a lowering in the concentration of DHA in tissues may in part be caused by a competitive inhibition with trans fatty acids for the incorporation into membrane phospholipids.

During the fetal and early postnatal development, the high requirements of LC-PUFA by the CNS and other tissues, as well as a further inhibition of their limited perinatal synthesis by trans fatty acids, could have serious consequences for neuronal and visual function. In humans, Koletzko (4) and Houwelingen and Hornstra (13) indicated an impairment of early growth by trans isomers in preterm and term neonates, whereas Opstvedt and Pettersen (29) did not find any impairment effect of dietary trans fatty acids on growth, nerve histology, or function in sows and offspring. Thus, more studies are needed to establish the possible physiologic repercussions of dietary trans fatty acids.

In conclusion, the results of the present experiment demonstrate that dietary trans fatty acids are incorporated into pregnant mother tissues in a dose-dependent manner, except for the brain, and that those fatty acids cross the placenta and are also incorporated into fetal liver but not fetal brain, provided that the diets contain enough amounts of EFA. A high intake of trans fatty acids substantially increases the plasma and liver linoleic acid concentrations as well as lowering DHA. In addition, the determination of delta-6 fatty-acid desaturase activity in the liver microsomes of the pregnant rats revealed inhibition by trans isomers.