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Sección de Nutrición y Bromatología, Departamento de Ciencias Biomédicas, Facultad de Ciencias Experimentales y Técnicas, Universidad San Pablo-CEU, 28668 Boadilla del Monte, Madrid, Spain
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • dietary folate • pregnancy • fat and protein utilization • toxicity • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, Czeizel et al. 1996
, Medical Research Council Vitamin Study Research Group 1991
). In addition, there is now agreement that good
folate status is associated with lower homocysteine levels, an amino
acid that was recently confirmed as a risk factor for vascular disease
(Loscalzo 1996
).
Because of these observations, national expert committees have reported
that at least 400 µg/d folic acid is necessary to maintain normal
folate status during pregnancy and that women who have already had a
neural tube defect birth should increase the dose up to 4 mg/d, which
means 20 times the established recommendation for nonpregnant woman
(200 µg/d) (Center for Disease Control 1991
,
Department of Health and Human Services 1992
,
Food and Drug Administration 1994
National Health
and Medical Research Council 1992
). More recently, new Dietary
Reference Intakes for folate were reported (Bailey 1998
). The concept of Dietary Folate Equivalents was introduced
to differentiate, for the first time, between food folate and synthetic
folate (used for supplementation and fortification) based on
differences in bioavailability, and the recommendation for pregnancy
has been raised to 600 µg/d Dietary Folate Equivalents (Bailey 1998
).
In addition to recommending supplement use, it has been suggested that
food fortification with folic acid could lead to an adequate folate
status for all women capable to become pregnant (Center for Disease Control 1994
, Food and Drug Administration
1993
, Health Council/Food and Nutrition Council
1992
, United Kingdom Department of Health 1992
).
Folic acid was traditionally considered safe because of its
water-soluble character (Butterworth and Tamura
1989
). However, it is necessary to question if extended
supplementation with doses between 0.4–4 mg/d or food fortification is
associated with adverse effects.
The most well known risk of exposure to high doses of folic acid is the
possible masking of cobalamin deficiency in pernicious anemia
(Lachance 1998
) because folic acid supplementation may
improve hematological indices but not the neurological disease of a
prolonged cobalamin deficiency. This was in fact the critical point for
estimating the Tolerable Upper Intake Level for folate, set at 1 mg/d
folic acid for all adults, including pregnant and lactating women
(Bailey 1998
). However, few studies on the effects of
acute or long-term, high doses of folic acid have been carried out
(Scott et al. 1997
), mostly because of ethical reasons,
so we do not have enough information to confirm the potential safety of
this vitamin per se or on its interrelationships with either macro or
micro nutrients. Hence, although folic acid is generally well
tolerated, supplementation could induce adverse effects we are unaware
of at present. This could be important in a physiological situation of
nutritional stress such as pregnancy, with possible
nutrient-nutrient or nutrient-drug interactions.
On the basis of all these issues, this study was undertaken to determine the effects of long-term dietary folate supplementation on several gestational and nutritional markers during pregnancy in rats, compared to a nonpregnant controls.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Thirty eight female Wistar rats (initial weight of ~180 g; Animal Service, Universidad San Pablo-CEU, Madrid, Spain) were classified into four groups on the basis of physiological status and the experimental diet administered: 1) dams fed the folic acid-supplemented diet (40 mg folic acid/kg diet); 2) dams fed the control diet (2 mg folic acid/kg diet); 3) virgin rats fed the folic acid-supplemented diet; and finally, 4) virgin rats fed the control diet. Animals were individually housed in metabolic cages specially designed for pregnant rats and were maintained in a room with a 12 h light/dark cycle, at 20–23°C, and with an appropriate ventilation system.
The folic acid concentration in the control diet was 2 mg/kg, adequate
for Wistar pregnant rats (Alonso-Aperte 1997
), whereas
40 mg/kg folic acid was the concentration employed in the supplemented
diet. The composition of both diets was similar, except for the above
mentioned differences in folic acid concentration. Both diets were
adjusted to rat requirements (National Research Council 1995
) and were based on the pure amino acid diet (17% amino
acid, Dyets, Bethlehem, PA) described by Walzem and Clifford (1988)
.
This is the most reliable design for studying the exclusive effect of
dietary folic acid, without confounding factors, as we have
demonstrated in several studies (Alonso-Aperte and Varela-Moreiras 1996
, Varela-Moreiras and Selhub 1992
, Varela- Moreiras et al. 1995
).
All experiments were undertaken according to Directional Guides Related
to Animal Housing and Care, from the European Community Council (1986)
.
Experimental design.
Pregnant and virgin rats were fed their respective folic acid-supplemented or control diet for 3 wk, corresponding to a complete pregnancy. All groups had free access to food and water. Dietary intake and body weight were assessed every 48–72 h, and the food efficiency ratio (FER)2 was calculated. Two critical periods were selected to carry out metabolic balance studies: d 1–5 and 17–21 of the experiment, which allowed us to determine fat and protein digestibilities as well as metabolic protein utilization. Urine and feces were collected daily during these two periods and stored at -20°C for further protein and fat determinations. On day 21 of the experiment, rats were anesthetized with CO2 and killed by decapitation. Embryonic development was evaluated by measuring fetal length and weight and total number of resorptions.
Fat determination.
Fat was determined in feces according to the Soxhlet method, using a Soxtec System (model 1043, Tecator, Höganäs, Sweden), and then the fat digestibility coefficient (FDC)2 was calculated as (fat intake - fecal fat)/fat intake.
Nitrogen determination.
Nitrogen was determined in urine and feces according to the Kjeldahl
method using a Kjeltec System (model 2006, Tecator). Therefore, the
following coefficients were calculated:
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Differences in means were studied by two-way ANOVA. When ANOVA resulted in differences, multiple comparisons between means were studied by the Tukey test. A paired Student's t-test was used to evaluate the differences in the same group between metabolic balance periods. All values are expressed as means ± SEM. Differences were considered significant at P < 0.05. (SYSTAT Version 5.0, Systat, Chicago, IL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Folic acid supplementation did not alter dietary intake (Fig. 1
) or body weight (Fig. 2
) significantly. Pregnancy, regardless of dietary folic acid level,
resulted in a significantly higher (P < 0.01) FER
(Table 1
) compared to virgin groups as a direct consequence of a greater
body weight gain (P < 0.001) (Fig. 2)
.
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On the basis of the number of live fetuses, gestational development was
considered adequate in both groups (Table 2
). However, body weight and vertex-coccyx length were
significantly lower (P < 0.001) in fetuses from
supplemented dams compared to those from control dams.
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FDC was not altered either by supplementation or by pregnancy, with
values that were satisfactory (Table 3). Moreover, there was no difference between the two metabolic
balance periods (d 1–5 and 17–21) because some small differences in
fat intake and fecal fat (Table 3)
, did not affect the final
coefficient.
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). However, the NDC was significantly lower in both groups of dams
during the second metabolic period compared to control virgins and to
the first metabolic term. This effect is a clear consequence of a
greater elimination of fecal nitrogen (P < 0.05)
(Table 4)
during late gestation.
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. However, folic acid supplementation significantly
reduced (P < 0.05) the MPU coefficient during the
second metabolic period (ds 17–21) (Table 4)
, an effect that was most
dramatic in virgin rats. As expected, a similar pattern was observed
for NPU: folic acid supplementation either in dams or virgin rats
resulted in a significantly lower (P < 0.05) NPU
coefficient than in control rats (Table 4)
. Urinary nitrogen was also
significantly higher (P < 0.05) in virgin rats fed the
folic acid-supplemented diet during the second metabolic period
(Table 4)
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The highest folate recommendation is that of 4 mg/d, a dose recommended
to prevent neural tube defect recurrence (Center for Diseases
Control 1991
). This is almost twenty times the 180–200 µg/d
recommended for nonpregnant women (Health Council/Food and
Nutrition Council 1989
, Moreiras et al. 1998
, National Research Council 1989
,
United Kingdom Department of Health 1992
), although this
was recently revised, and now 400 µg/d is the amount considered
adequate for women (Bailey 1998
). On this basis, this
study was conducted to examine the effects of long-term extra folic
acid dietary supplementation (20-fold the rat requirement) on several
gestational and nutritional markers compared to a nonpregnant controls.
When general nutritional status was studied, we observed that folate
supplementation did not affect growth in rats, which was consistent
with other studies in which folate rich diets do not improve growth
response (Alonso-Aperte 1997
, Clifford et al. 1989
) However, as expected, pregnancy resulted in a higher FER
compared to virgin groups, indicating an adequate gestational
development for dams fed both folic acid levels. Gestational
development was also considered adequate in both groups based on the
number of live fetuses. Despite this, there were a significant
reductions in body weight and vertex-coccyx length in fetuses from
dams supplemented with dietary folic acid. The consequences of these
interesting observations are unknown at present, and it would be useful
to study the postpartum development of these low weight and length
newborn fetuses. It is worth mentioning, regarding this issue, that
growth and cellular division are among the main functions of folic
acid. For this reason supplemental folate is being recommended to all
pregnant woman during the second trimester of gestation, alone or in a
multivitamin complex. This goal, however, is very different from the
recommendation of high or even pharmacological doses recently
considered to prevent neural tube defects. To our knowledge, our data
are the first to show these results.
Because there is a lack of information regarding potential
micro-macro nutrient interactions, we questioned whether high folic
acid intake may result in alterations of general digestive function.
The dietary FDC is a critical marker to determine whether diet
utilization is affected. No differences were observed in any of the two
metabolic balance studies; the values obtained were excellent and in
the range of those obtained in other metabolic studies evaluating
potential drug-nutrient interactions (Ragel et al. 1994
, Varela-Moreiras et al. 1991
).
Folic acid supplementation also did not alter NDC. Conversely,gestation itself influenced nitrogen absorption. During late gestation, fecal nitrogen elimination was greater than during early gestation, an effect that was independent of folic acid supplementation and that we, therefore, consider to be physiological.
MPU was significantly lower in folate-supplemented groups during the second metabolic period (d 17–21). This observation may be of critical importance because from our studies it may be clearly hypothesized that supranormal folate intakes are associated with an impairment in dietary protein metabolic utilization. This unexplained effect could be related to a xenobiotic behavior of folic acid when it is consumed at high levels.
Like MPU, NPU, a useful index for evaluating both digestive and
metabolic protein utilization, was also lower in supplemented groups
compared to controls. This effect was most marked in virgin rats.
Because nitrogen intake and digestibility in the virgins were not
changed throughout the study, the reduction in MPU and NPU can be
explained only as a consequence of a significantly higher urinary
nitrogen elimination (Table 4)
.
Pregnant rats also showed lower MPU and NPU values after long-term folic acid supplementation, although in this group the effect cannot be explained as a consequence of a higher nitrogen urinary elimination. Because pregnancy itself seems to influence NDC, the effect of folic acid supplementation on MPU and NPU is more complicated.
Long-term folic acid supplementation at doses 20-fold higher than the rat requirements may alter nutrient metabolic utilization without affecting digestive function. During gestation, this effect may be associated with reduced fetal growth. Future studies will be necessary to further develop or refute these conclusions.
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FOOTNOTES |
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Manuscript received September 11, 1998. Initial review completed October 26, 1998. Revision accepted February 3, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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