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*
Department of Physiology and Pharmacology, University of Murcia, Murcia, Spain; and
Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-linolenic acids and
a ratio of 18:2(n-6)/18:3(n-3) of about 7:1. Trans fatty
acids were incorporated into maternal milk in a dose-dependent
manner. In addition, rats fed trans isomers had greater
linoleic acid levels than controls. The proportion of -linolenic
acid in milk was lower in the VH group, and the (n-6)/(n-3)
cis PUFA ratio in milk of the VH group was greater than
that in controls. Total long-chain PUFA levels did not differ among
groups. These results suggest that high intakes of trans
fatty acids affect the EFA concentration but not that of long-chain
PUFA of rat milk, provided that EFA are supplied in sufficient
amounts.
KEY WORDS: • trans fatty acids • rats • milk • EFA • PUFA
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Dietary trans
fatty acids influence the lipid composition of biological fluids,
namely milk (Chen et al. 1995
) and serum (Judd et al. 1994
), as well as tissues in humans (Ohlrogge 1979
) and animals (Grandgirard et al. 1994
).
Although trans fatty acids might interfere with the
biosynthesis of long-chain PUFA
(LC-PUFA)2
(Houwelingen and Hornstra 1994
) and intensify the
biochemical and physiological alterations of essential-fatty acid
(EFA) deficiency (Beyers and Emken 1991
), only a few
studies have investigated the biological impact of dietary
trans fatty acids during infancy. Nevertheless, the general
consensus is to moderate the consumption of these fatty acids due to
their potential adverse effects throughout childhood (Stender et al. 1995
, Wahle and James 1993
).
In recent years, increasing attention has been focused on determining
the distribution and concentration of trans fatty acids, due
to the importance of lipids in infant growth and development.
Trans fatty acid milk composition varies depending on
seasons and regions, and different feeding practices (Jensen and Lammi-Keefe 1998
). Studies on human milk in different
countries have shown marked differences in trans fatty acid
concentration, varying from 7.2% in Canada (Chen et al. 1995
), with a pattern of trans fatty acids similar
to that of partially hydrogenated oils used in the mother’s diet, to
1.9% in France (Chardigny et al. 1995
), vaccenic acid
being the predominant isomer, suggesting the influence of cow’s milk
fat in the diet. In general, the trans fatty acid
concentration of milk reflects trans fatty acid intake.
Petersen and Opstvedt (1991)
have studied the influence of high levels
of dietary trans fatty acids with a parallel drop in
saturated fatty acids (SFA) on the lipid composition of colostrum and
milk in pigs, concluding that changes in the fat concentration and the
fatty acid composition of colostrum and milk in sows were moderate to
minor and that no consistent effects on the levels of polyenoic fatty
acids were evident. However, other authors have reported high levels of
polyenoic isomers in the milk of rats fed partially hydrogenated marine
oil (Brandorf 1996
).
Insufficient data are available to assess the effects of dietary
trans fatty acids on maternal milk, and controlled studies
are needed to establish whether there is a dose-response
relationship and whether trans fatty acids could affect the
concentration of EFA, LC-PUFA and the (n-6)/(n-3) ratio in milk.
Thus, the aim of the present study was to evaluate the effects that
three diets, differing mainly in their trans fatty acid
levels but containing the same proportion of linoleic [18:2 (n-6)],
-linolenic [18:3 (n-3)] acids, exert on the fatty acid profile of
milk rat, placing particular emphasis on the distribution and
concentration of trans isomers, EFA and LC-PUFA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The protocol of this study was approved by the Animal Laboratory
Service of the University of Murcia (Murcia, Spain), and animals
received humane treatment according to the regulations for Animal
Research of the European Union. Female Wistar rats at weaning
(21-d-old), supplied by the Animal Laboratory Service of the University
of Murcia, were kept individually in metabolic stainless steel cages in
a room with controlled temperature (22°C) and light (0800–2000 h)
and allowed free access to food and water. The rats were assigned to
three groups of six, matched by mean weight. Rats were fed for 10 wk
until milk sampling on three isocaloric
diets3
(1743 kJ/100 g) that differed in their fatty acid profile. The
fat (per kilogram of diet) consisted of a mixture of 75 g olive
oil and 25 g soy oil for the diet very low in trans
fatty acid concentration (Control diet, total trans
concentration of about 0 mol/100 mol); 37.5 g olive oil, 34 g
vegetable shortening and 28.5 g soy oil for the high
trans group (H diet, total trans
concentration of about 14.5 mol/100 mol); and 68 g vegetable
shortening and 32 g soy oil for the group very high in
trans concentration (diet VH, total trans
concentration of about 30 mol/100 mol). The diet classification was
established on the basis of the trans concentration in
the human diets. The fatty acid composition of the three diets is
summarized in Table 1
. The diets were prepared using the same batch of raw materials and
stored at -4°C.
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After 7 wk of consuming their respective diets, female rats were mated
(1:1 in each cage), and on d 1 of pregnancy, as indicated by the
presence of a mating plug, male rats were separated and female rats
remained in their cages throughout the pregnancy period (3 wk). Six
dams were in each experimental group, and on d 3 postpartum, four pups
per dam rat were killed with sodium pentothal and the total contents of
their stomachs were pooled and taken as curd-milk for each dam,
frozen in liquid nitrogen and stored at -80°C until analysis of the
milk-curd fatty acid composition, as previously described
(Sanders et al. 1984
).
Analytical methods.
Total lipids from rat curd-milk were extracted with
chloroform/methanol (2:1, v/v) according to the method of Folch et al. (1957)
, and fatty acid methyl esters (FAME) were prepared with
methanolic HCl 3 mol/L (Supelco, Bellefonte, PA) at 85°C for 1 h
and dissolved in hexane. FAME were analyzed by gas- liquid
chromatography (GLC), using a SP-2560 flexible fused silica capillary
column (100 m x 0.25 mm i.d., 20 µm film thickness; Supelco) in a
Hewlett-Packard 5890-A gas chromatograph. The oven temperature was
programmed 39 min at an initial temperature of 175°C and was
increased at a rate of 3°C per min to 230°C and held at that
temperature for 14 min. The injector and detector were set at 250°C.
Helium was used as a carrier gas at a pressure of 290 kPa, and peaks
were identified by comparison of their retention times with appropriate
FAME standards purchased from Sigma Chemical Company (St. Louis, MO).
The double-bond positions of isomers were confirmed by GLC/mass
spectrometry (GLC/MS) analysis of their 2-alkenyl-4,4-dimethyloxazoline
(DMOX) derivatives, prepared as previously described (Luthria and Sprecher 1993
). For GLC/MS analysis of the DMOX, a Varian
GC Analytical MS System (Model 7070EQ) was used; the system was
equipped with an 11/250 data module (Model Vista 6000) and operated at
an ionization energy of 70 eV. Gas chromatography separation of the
DMOX derivatives was performed on the same SP-2560 capillary column
described for the FAME analysis, and helium was also used as a 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 separation and quantification of 18:1 isomers in the diets and
rat milk and to avoid the overlap of 18:1(n-6)t,
18:1(n-5)t and 18:1(n-4)t with 18:1
cis isomer peaks, FAME were fractionated using silver
nitrate thin-layer chromatography (AgNO3-TLC) and
capillary GLC, following the procedure of Ulberth and Henninger (1992)
.
Statistical methods.
Results were expressed as mean proportions (mol/100 mol total fatty
acid esters) ± SEM. Since fatty acid molar
percentages usually are not normally distributed, data were transformed
to their decimal logarithms. One-way ANOVA was used to assess the
effect of dietary trans fatty acid on milk fatty acids
using the Statgraphics 7.0 (Statistical Graphics, Bitstream, Cambridge,
MA) software. Bonferroni’s multiple range test was performed to
identify differences between groups at = 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
|
.
However, all 16:1 and 18:1 trans isomer levels were higher
in the milk of dams fed trans fatty acid diets (Table 2)
,
and levels were directly related to the H and VH dietary
trans fatty acid concentrations (r = 0.969,
P < 0.001). This resulted in a higher proportion of
trans MUFA in the milk of rats fed the trans
diets than in controls (Fig. 1
). On the contrary, milk cis MUFA were significantly lower
than controls only in the VH group (Fig. 1)
. The major difference noted
in these latter fatty acids was the low proportion of 18:1(n-9) in the
VH group (Table 2)
. Positively correlated was 18:1(n-9) cis
(r = 0.89, P < 0.0001) with the
dietary 18:1 (n-9) concentration and inversely proportional
(r = -0.896, P < 0.0001) to the
trans fatty acid concentration in the experimental diets.
Other positional 18:1 cis isomers rose in parallel with the
concentration of these fatty acids in the H and VH diets (Table 2)
.
|
. Levels of
trans isomers of 18:2 and 18:3 fatty acids rose parallel
with their concentrations in the diet (Table 2)
. The proportion of
18:2(n-6) also rose significantly, parallel with the total
trans concentration in the experimental diets (r
= 0.85, P < 0.0001). On the contrary, the levels
of 18:3(n-3) were lower only in the VH group. Cis (n-6) PUFA
were significantly higher in the H and VH groups than in the controls
(Fig. 1)
, due mainly to the greater proportion of 18:2(n-6) (Table 2)
.
This led to a significantly greater (n-6)/(n-3) ratio in the VH group
than in the other two groups (Fig. 1)
. However, no significant
differences were found for total or individual LC-PUFA [i.e.,
20:4(n-6), 20:5(n-3) and 22:6(n-3)], among the experimental groups
(Table 2)
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and term neonates (Houwelingen and Hornstra 1994
), and the potential negative effects on EFA
metabolism have caused concern about the exposure of infants to
trans fatty acids. In different countries, studies have
examined the concentrations of these fatty acids in human milk
(Chen et al. 1995
, Koletzko et al. 1992
).
However, the individual diet of the mother can alter the fatty acid
profile in milk, and different diets could be consumed even in a
geographically small area, hampering general conclusions from being
drawn about the trans fatty acid concentration in milk.
Thus, Jensen et al. (1992)
have emphasized the need for more data in
order to evaluate dietary effects on fatty acids in milk, using the
best available analytical methods. The analytical method we used is very sensitive and highly reproducible, allowing us to make precise determinations of dietary and milk trans fatty acids. The use of Ag-TLC and long GLC capillary columns, in addition to standardized trans fatty acids as well as previous data on GLC-MS of those fatty acids, enabled us to quantify and to determine the position and geometry of double bonds in diet and milk fatty acids.
The results of the present study confirm a direct dose-response relationship between the intake of trans fatty acids and their incorporation in maternal milk. Thus, the trans fatty acid profile of milk fat was quite similar to that of the vegetable shortening used as source of trans isomers in the experimental diets, indicating that maternal milk may contain high levels of trans fatty acids.
Bovine milk used in infant formulas contains trans fatty
acids from the diet or bacterial activity in the rumen. Their
concentrations of trans fatty acids in human milk depend on
dietary lipids (Wonsil et al. 1994
). Craig-Schmidt et al. (1984)
developed an equation to correlate the percentages of
18:1 trans in human milk [y] and maternal diet [x]) [y
= 1.49 + 0.42x]. This equation predicts human milk 18:1
trans percentages close to that found in the present study
for rat milk using [y = 0.33 + 0.42x].
SFA in the three diets differed, but in contrast to the report of
Pettersen and Opstvedt (1991)
, in the present study, trans
fatty acids substituted mainly for cis fatty acids and were
incorporated in diets H and VH to study the effect of trans
respect to cis geometry. MUFA levels in milk depend, to a
certain extent, on the dietary intake of these fatty acids
(Koletzko et al. 1992
). The milk total MUFA did not
differ among groups as occurred in the diets, but significant
differences in the milk cis and trans MUFA fatty
acids among experimental groups were found. Nevertheless, whether
trans fatty acids directly alter the cis fatty
acid incorporation in rat milk could not be ascertained in the present
study because the former fatty acids constituted the main variable
which enabled us to adjust the diets.
The results of the present study showed a considerable rise in the
levels of 18:2(n-6) in rat milk, directly related to the
trans fatty acid intake. However, arachidonic acid (AA),
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) proportions
in milk were not affected by dietary trans fatty acid
concentration. Chen et al. (1995)
found in an epidemiological study an
inverse correlation between trans fatty acids and the levels
of 18:2(n-6) and 18:3(n-3) in Canadian human milk (r = -0.29, P < 0.0001; r = -0.25,
P < 0.001), suggesting that the rise in
trans fatty acids may occur at the expense of EFA, since
high trans dietary products usually contain less of these
last fatty acids. On the contrary, Petersen and Opstvedt (1991)
showed
an increase in 18:2(n-6) in milk from sows fed partially hydrogenated
soybean oil vs. lard. Our results agree with this latter study, and one
of the hypotheses to explain the increase of 18:2(n-6) in the milk of
rats fed high trans fatty acid diets is that the mammary
gland might regulate the acylation of milk triglycerides and
phospholipids in order to maintain the physicochemical properties of
the fat globules. Our data support this hypothesis, since the
unsaturation index (defined as 
molar proportion of fatty acids per
number of cis double bounds) of milk was fairly constant
regardless of the trans fatty acid concentration (67.16
± 2.22 mol/100 mol, 70.26 ± 4.77 mol/100 mol, 61.07 ± 3.22 mol/100 mol, for control, H and VH groups, respectively).
Some authors have suggested that trans fatty acids may
inhibit tissue 
6 fatty acid desaturase (Cook and Emken 1990
, Sugano and Ikeda 1996
) which would lead to
higher levels of 18:2(n-6) in body fluids. If this assumption is true,
then LC-PUFA levels, namely AA, EPA and DHA, would fall. However,
in the present study, LC-PUFA remained nearly constant in milk.
Gibson and Kneebone (1984)
demonstrated the lack of correlation between
LC-PUFA and their respective fatty acid precursors in human breast
milk. Milk AA is affected little by diets as different as those heavily
omnivorous and those basically vegetarian (Sanders and Reddy 1992
), or African and European (Koletzko et al. 1992
), suggesting the presence of a protective mechanism for
the infant by providing a relative and constant dietary supply of
LC-PUFA.
The assimilation of appropriate amounts of (n-6) and (n-3) fatty acids
in tissues of growing infants depends of their administration in
maternal milk. In this study, dietary trans fatty acids
modified the (n-6)/(n-3) ratio in milk from rats fed higher proportions
of trans isomers in diet, and no studies have found that
trans fatty acids might alter this ratio. The experimental
studies of Petersen and Opstvedt (1991)
did not consider the
linoleic/-linolenic acid ratio in milk, and they used diets with a
percentage of energy as EFA much higher than used in this study (about
8% energy vs. 4% energy) which might affect to the metabolic pathways
of these fatty acids. Teter et al. (1992)
used levels of EFA similar to
this study, but they did not make reference to the influence of dietary
trans fatty acids on the (n-6)/(n-3) ratio in milk of mice.
In summary, the results of our study demonstrate that trans fatty acids are incorporated into maternal milk in a dose-dependent manner and that they may accumulate in high levels when the dietary concentration is also high. In addition, trans isomers may modify the level of EFA and increase the (n-6)/(n-3) ratio in milk but does not affect the total LC-PUFA levels, provided that sufficient amounts of dietary EFA are supplied.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Composition of diets: Each kilogram of diet contained; 190 g casein, 10 g DL-methionine,
100 g fat mixture, 100 g sucrose, 418 g starch, 80 g cellulose, 2 g choline bitartrate, 50 g mineral supplement
and 50 g vitamin supplement. Composition of the mineral and
vitamin supplements complied with the AIN-93 recommendations (Reeves et al. 1993
).
Manuscript received May 11, 1999. Initial review completed July 16, 1999. Revision accepted December 8, 1999.
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