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Department of Health Studies and Gerontology, University of Waterloo, Waterloo, ON, Canada N2L 3G1
3To whom correspondence and reprint requests should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • EFA deficiency • zinc deficiency • arachidonic acid • docosahexanoic acid • brain development • artificially reared rat pups
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Linoleic acid
[LA,4
18:2(n-6)] and linolenic acid [LNA, 18:3(n-3)] are necessary for
normal growth and development (Innis 1991
). They are
called essential fatty acids (EFA) because they cannot be synthesized
de novo by animals and must be provided by the diet. These 18-carbon
fatty acids are converted to long-chain polyunsaturated fatty
acids (LCPUFA) through a series of desaturation and elongation
reactions (Cook 1991
). EFA and their longer-chain
metabolic products are structurally important as components of membrane
phospholipids, and the 20-carbon compounds are functionally important
because they are precursors in the formation of the eicosanoids
(prostaglandin, thromboxanes and leukotrienes).The prostaglandins are
important contributors to regulatory functions in the brain, and they
can influence neural activity indirectly by modulating neurohormones
and neurotransmitters (Wainwright 1997
). Dietary fatty
acids will change the fatty acid composition of membrane phospholipids
in the developing central nervous system (Innis 1994
)
and thus may potentially influence brain functions. EFA deficiency in
animals has been shown to contribute to aberrations in cognitive
development [reviewed by Wainwright (1992)
].
Zinc is an important trace element for normal cell growth and
development in mammalian species. Zinc plays many diverse roles in cell
biochemistry, and zinc deficiency has been associated with an array of
symptoms such as delayed growth and wound healing, acrodermatitis,
delayed sexual maturation as well as abnormal behavioral development
(Prasad 1988
). During development, zinc is important for
normal morphogenesis of the central nervous system, and it appears to
play a functional role in regulating the release of neurotransmitters
such as 
-amino butyric acid, acetylcholine and glutamate
(Dreosti 1993
, Prasad 1997
). The presence
of high concentrations of zinc in the hippocampus (Pfeiffer and Braverman 1982
), together with its function in biochemical
processes in the brain may relate to observations of impaired learning,
reduced activity and poorer memory reported in several studies in
zinc-deficient animals [summarized by Golub et al. (1995)
].
Analogies between the pathology of EFA deficiency and that of zinc
deficiency were first described in rats by Bettger and co-workers (1979)
. These investigators demonstrated that
zinc deficiency intensified the effects of EFA deficiency; thus, an
interaction between zinc and EFA metabolism was proposed. It has been
demonstrated in several animal studies that dietary zinc deficiency
alters the fatty acid composition of phospholipids of the liver and red
blood cells (Bettger et al. 1979
, Clejan et al. 1982
, Cunnane 1988
, Eder and Kirchgessner 1994a
and 1994b
, Kudo et al. 1990
). The findings
of these studies, however, have been contradictory. For example, some
studies reported higher levels of LA and lower levels of arachidonic
acid [AA, 20:4(n-6)] in tissue phospholipids of zinc-deficient
animals (Clejan et al. 1982
, Cunnane 1988
). These findings suggested a role of zinc in 
5- and
6-desaturase enzyme activity, and a reduction of these enzyme
activities has been described in zinc-deficient animals
(Clejan et al. 1982
). However, findings from other
studies do not support this hypothesis (Eder and Kirchgessner 1994a
and 1994b
, Kudo et al. 1990
). Eder and Kirchgessner (1994b)
, found greater levels of (n-3) LCPUFA
in liver phospholipids of zinc-deficient young adult rats and
Kudo et al. (1990)
found greater AA levels in liver and
plasma phospholipids of zinc-deficient adult rats. In this latter
study, however, a fat-free diet was used.
The contradictory results of these studies may be explained by the
reduced food intake resulting from zinc deficiency that was observed in
some of these studies (Clejan et al. 1982
, Kudo et al. 1990
). This in itself may affect EFA metabolism
(Kramer et al. 1984
). Furthermore, Eder and Kirchgessner (1994b)
demonstrated that the type of dietary fat
influences the effects of zinc deficiency on fatty acid composition of
liver lipids. In this study, it was found that when zinc-deficient
rats were fed a coconut oil–based diet, lower levels of AA were
replaced by docosahexaenoic [DHA, 22:6(n-3)] and docosapentaenoic
acid [22:5(n-3)], whereas when they were fed a fish oil diet, AA was
replaced by eicosapentaenoic acid [20:5(n-3)]. Thus the variability
among studies in terms of dietary fatty acid composition is also a
possible factor contributing to the contradictory findings.
Regardless of the exact mechanisms involved, zinc deficiency does
appear to affect EFA metabolism and results in altered LCPUFA
composition of tissue lipids. This could have important consequences
for brain development, particularly during rapid brain growth. This
issue may be of particular importance to preterm infants, who miss the
period of peak accumulation of body zinc stores as well as the
accretion of LCPUFA in the brain during the last trimester of pregnancy
(Innis 1991
, Zlotkin and Cherian 1988
).
An adequate postnatal supply of zinc and EFA is critical to both
restore and maintain zinc stores and support the rapid accumulation of
LCPUFA, respectively, in the brain. Several studies have demonstrated
that preterm infants benefit from additional dietary zinc with regard
to locomotor development (Friel et al. 1993
) and also
from dietary LCPUFA with regard to visual acuity (Birch et al. 1992
, Carlson and Werkman 1996
). Findings from
such clinical trials suggest that preterm infants may experience
suboptimal zinc as well as EFA status. Because of the importance of
both zinc and EFA for brain and behavioral development and the role
that zinc may play in EFA metabolism, the objective of this study was
to investigate the interactive effects of zinc and EFA in a study
design relevant to infant nutrition.
The animal model used for this purpose was the artificial rearing model
for neonatal rats, in which infant rats are reared independently of
their mothers via gastrostomy tubes (Hall 1975
). The
optimal time during which to investigate the interactive effects of
zinc and EFA on brain development is when the brain is growing most
rapidly, i.e., the brain growth spurt. In the rat, which is the most
commonly used animal model in behavioral science, the period between
birth and weaning (at ~d 18–22 postnatal age) is equivalent to the
human brain growth spurt, which occurs between the third trimester and
the age of 2 y (Dobbing 1975
). Thus the artificial
model allows direct manipulation of the diet during this sensitive
period. It also avoids the problem of reduced voluntary food intake
encountered in conventional zinc deficiency experiments. In adult
animals, these problems have been overcome by feeding the animal via
gastric tubes (Eder and Kirchgessner 1994a
and 1994b
,
Yang and Cunnane 1994
). However, when measuring effects
of nutrition during the early neonatal period on brain and behavioral
development, usually one has to manipulate the diet of the mother to
alter nutrient intake in her offspring. With this practice, changes in
maternal physiology and/or behavior due to the nutritional condition of
the mother may affect the offspring indirectly and become a confounding
variable. Although the artificial rearing model circumvents this, it is
important that such studies include a normally suckled control group
for comparison, particularly when the outcomes being measured include
behavioral development. In this study, a 2 x 2 design was used to
manipulate dietary zinc and EFA composition, and a fifth suckled
control group was included. The intent was to determine whether, in an
appropriate animal model, short-term dietary zinc deficiency during
the early neonatal period exacerbates the effects of EFA deficiency on
brain and liver LCPUFA composition, as well as on behavioral
development
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male offspring of timed-pregnant Long-Evans rats (Harlan Sprague
Dawley, Indianapolis, IN) were used. Pregnant dams were obtained at d
10–14 of gestation and were housed individually with free access to
AIN-93M nonpurified diet (Reeves et al. 1993
) (Dyets,
Bethlehem, PA). They were maintained at 22 ± 1°C under a
reversed 12-h light:dark cycle. Litters were culled, when necessary, to
10 pups within 12 h of birth. Male pups from each litter were
assigned randomly to the diet groups, with no more than one pup from
any litter being assigned to a single diet group. The sample size
ranged from 9 to 14 animals per group for locomotor skills testing and
from 8 to 13 for tissue chemistry. 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 composition of the rat formula has been described in detail
previously (Ward et al. 1998
). The zinc concentration of
the zinc-deficient diet (Zn-def) was 39.8 ± 3.1
µmol/L; in rodents, this constitutes
moderate-to-severe zinc deprivation (Golub et al. 1995
).
The zinc concentration of the zinc adequate (Zn-adeq) diet was 468.6
± 104.1 µmol/L. This level was chosen to be
somewhat higher than that of rat milk (~300–380
µmol/L) to account for the lower bioavailability of
zinc from artificial formulas (Sandstrom et al. 1983
).
The dietary oils consisted of mixtures of medium-chain triglyceride
oil, coconut oil, soy oil and olive oil to obtain an EFA-deficient
(EFA-def) and an EFA-adequate (EFA-adeq) fat mixture. The
EFA-def diet contained marginal levels of LA and LNA to prevent
growth retardation and was therefore not completely deficient. The
fatty acid composition of the respective diets is shown in Table 1
. The different formulas were stored frozen under nitrogen. The diets
were coded with the intention that the experimenters would not be aware
of the dietary treatment to which each rat was assigned. However,
because the rats in the Zn-def group lost hair, the investigators
were aware of which had received the zinc-deficient diet.
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Four diet groups were used in a 2 x 2 design. These were
EFA-adeq/Zn-adeq, EFA-adeq/Zn-def, EFA-def/Zn-adeq and EFA-def/Zn-def.
A fifth group, the suckled control group, consisted of pups that were
fostered, on the day that their littermates were gastrostomized, to
nursing dams receiving AIN-93M diet (Reeves et al. 1993
).
Artificial rearing procedure.
This procedure has been described previously (Ward et al. 1998
). Briefly, on postnatal d 5, the rat pups were
anesthetized with methoxyflurane inhalant (Metofane, Janssen
Pharmaceutica, North York, Canada) and the gastrostomy tube was
inserted. The gastrostomy tube (Intramedic tubing, PE 10, Clay Adams,
Parsippany, NJ) has a small plastic flange at one end. The tube, which
was attached to a short wire contained within silastic tubing and
lubricated with medium-chain triglyceride oil, was inserted into
the mouth of the pup, down the esophagus and out through the stomach
wall. The survival rate of the gastrostomy procedure was ~90%. Pups
were housed individually in plastic cups floating in a water bath
maintained at 35 ± 1°C and were fed one of the experimental
diets via polyethylene tubing (Intramedic tubing, PE 10, Clay Adams)
attached at one end to their gastrostomy tubes and at the other end to
syringes attached to an infusion pump (Model 55–4143, Harvard
Apparatus, South Natick, MA). The pumps were programmed to deliver the
formula for 10 min every hour. The pups were fed an amount of diet that
represented 29% of their body weight (adjusted daily) at the start of
the study and was increased to 35% of body weight after ~5–6 d. All
pups including the suckled controls were handled daily. The pups were
weighed daily, their gastrostomy tubes were flushed with 0.1 mL of
distilled deionized water, and their anogenital regions were washed
gently with a wet tissue to stimulate urination and defecation. Suckled
control pups were also weighed daily. Eye opening was checked twice
daily (morning and evening) from d 12 onward. At the end of the study
at d 16, the rats were anesthetized under Halothane (MCT
Pharmaceutical, Cambridge, Canada); when rats were completely
unconscious, 1 mL of blood was removed by cardiac puncture, and whole
brain (consisting of forebrain and cerebellum) and livers were removed
and weighed. The brain was cut sagitally into two equal portions, one
for fatty acid composition and one for elemental zinc analysis.
Similarly, the liver was divided in two portions. Plasma, liver and
brain samples were stored at -80°C until further analysis.
Zinc analysis.
Liver and brain samples were dried at 75°C overnight and weighed. Then the samples were ashed at 500°C (Thermolyne Furnace 304000, Sybron/Thermolyne Corporation, Dubuque, IW) and reconstituted in 1.6 mol/L nitric acid. Plasma samples were measured, dried and ashed similarly as described above. Formula samples were wet digested (MDS 2000, CEM microwave sample preparation system,Matthews,NC) with nitric acid. Total zinc in formula, plasma, brain and liver were determined by flame atomic absorptiometry (Varian Spectra, Georgetown, Canada).
Lipid analysis.
The lipids in tissue and formula samples were extracted in
chloroform/methanol (1:1) with the use of a method modified from
Bligh and Dyer (1959)
. These lipid extracts were then
separated into different phospholipid fractions by TLC on silica gel
plates, using a solvent system of hexane/diethyl ether/acetic acid
(80:20:1) The fatty acids in the phosphatidylcholine (PC) and
phosphatidylethanolamine (PE) fractions were then esterified with 0.64
mol/L methanolic sulfuric acid and analyzed on a gas chromatograph
(Shimadzu GC-17A Gas Chromatograph, Shimazu Corporation, 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
150oC for 2 min, then programmed to 210°C at
3oC/min and held for another 10 min. The injector and
detector temperatures were maintained at 250oC. Fatty acids
were identified via comparison of retention times with authentic
standard mixtures (Nu-Chek Prep, Elysian, MN).
Testing of locomotor skills.
The pups were tested for locomotor development (hind-limb support while
suspended) and development of complex locomotor skills (ascending a
wire-mesh surface and traversing a narrow bridge). These tasks,
which are described below, were adapted from the methods described by
Altman and Sudarshan (1975)
. A pilot study was performed
initially for each test to determine the optimal day of testing for
Long-Evans rat pups. Optimal days of testing were as follows:
hind-limb support while suspended, d 16; ascending wire-mesh
surface, d 14; and traversing narrow bridge, d 16. Artificially reared
pups were placed in a cage together with litter mates of the suckled
control pups for 0.5 h before the behavioral testing. This was
done in order to stimulate homing in the artificially reared pups.
Hind-limb support while suspended. The synergistic support provided by the hind limbs to prevent falls or to aid progression along a wire was tested on d 16. A 2-mm thick, 60-cm long rope was extended horizontally between two poles ~30 cm high. The rat was placed with its front paws on the wire; grasping usually ensued immediately. The task was videotaped and scored for overall performance as follows: 1.0, strong grasp with fore- and hind limbs; 0.9, strong grasp with forelimbs and fair use of hind limbs; 0.8, forelimb grasp is strong, and hind limb grasp is present but weak; 0.7, forelimb is strong, and hind limb grasp is present but minimal; 0.6, forelimb grasp is adequate, and hind limb(s) touch but no grasp; 0.5, forelimb grasp is adequate, and hind limbs do not touch but rat attempts; 0.4, forelimb grasp closes but easily loses grip, hind limb(s) do not touch and minimal attempts are made; 0.3, forelimb grasp closes, but easily loses grip and no attempts are made to use hind limbs; 0.0. no grasp with fore- or hind limbs.
Ascending a wire-mesh surface. The ability of the pups to ascend a wire-mesh surface using homing as motivation and cold water as an averse stimulus was tested on d 14. A wire-mesh surface consisting of 6-mm wire mesh attached to a wooden frame 45 cm high and 15 cm wide was placed at an angle of 70° with its top in contact with a platform and its base in water (15°C). As an incentive, litter mates of the suckled control pups were placed on the platform and the test rat was placed at the base of the wire mesh, with its tail in the water. The rat was given a maximum of 2 min to reach the platform. The task was videotaped and scored for overall performance as follows: 1.0, ascends promptly up wire mesh using fore- and hind limbs; 0.9, as 1.0 but proceeds with hesitation; 0.8, ascends wire mesh, hind limbs are used more for support than thrust; 0.7, as 0.8 but proceeds with hesitation; 0.6, ascends only a few centimeters with minimal use of hind limbs; 0.5, no use of hind limbs but pulls up with forelimbs and ascends a short distance; 0.4, holds onto wire mesh but does not ascend.
Traversing a narrow bridge. The ability of pups to traverse a narrow bridge using homing as motivation was tested on d 16. In this test, two elevated platforms were connected by plywood (60 x 3 cm) at a height of ~4 cm. The pup was placed on the right platform, whereas litter mates of the suckled control pups were placed on the left platform. The pups were given a maximum of 2 min to traverse the bridge. The task was videotaped and scored for overall performance as follows: 1.0, promptly ventures onto the bridge using both fore- and hind limbs for support; 0.9, as 1.0 but with some hesitation; 0.8, ventures onto bridge but uses hind limbs only partially (e.g., one hind limb grasps, other drags along); 0.7, as 0.8 but with some hesitation; 0.6, ventures onto bridge but minimal use of hind limbs; 0.5, ventures partly onto bridge but turns around or falls; 0.4, does not venture onto bridge.
Statistical analysis.
The main effects of zinc, EFA and the interaction between zinc and EFA
on outcome measures were determined by two-way ANOVA, using SAS
v.6.0.9 (SAS Institute, Cary, NC). A significant interaction was
interpreted by a subsequent simple-effects analysis, i.e.,
comparison of zinc effects at each level of EFA in the artificially
reared groups with the use of an a priori Student's t
test. The effect of artificial rearing on the outcome measurements was
determined by comparing the "physiologic control," which was the
diet group Zn-adeq/EFA-adeq, to the suckled control groups by using
either Student's t test or the Mann-Whitney-U test when
data were not normally distributed. Because of the nature of the data,
the overall performance scores on the locomotor tasks were analyzed by
a "robust" procedure for a two-way ANOVA in which the overall
performance scores were transformed to ranks (Conover and Iman 1976
). The level of significance for all tests was
P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Weight gain and body weight at end of the study were different among
groups (Table 2
). Poorer weight gain (P < 0.001) and lower body weight
(P < 0.001) by the end of the study were observed in
the Zn-def groups. There was no main effect of EFA nor an
interaction between zinc and EFA for weight gain and brain or liver
weight. Weight gain and brain weight were significantly greater in the
suckled control group compared with the artificially reared
"physiologic control" (P = 0.001 and P
= 0.001, respectively). Eye opening did not differ among all diet
groups (data not shown).
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Zn-def resulted in significantly lower plasma (P < 0.001), brain (P = 0.008) and liver zinc concentrations
(P < 0.001). There was no main effect of EFA nor an
interaction between zinc and EFA. Brain and liver zinc concentrations
were lower in the suckled control group compared with the artificially
reared "physiologic control" (P = 0.012 and
P = 0.001, respectively) (Fig. 1
).
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The effects of the dietary manipulations were more pronounced in the PC
fraction (Table 3
) than in the PE fraction (Table 4
) of liver phospholipids. In liver PC, EFA-def reduced AA and DHA
and elevated 22:5(n-6) levels. Interactions between EFA and zinc were
observed for AA and DHA; Zn-def reduced AA and DHA levels only when
fed with EFA-adeq, and increased AA (but not DHA) levels when fed
with EFA-def (Table 3)
. In liver PE, an interaction between EFA and
Zn was observed for AA; Zn-def increased AA levels when fed with
EFA-def. Both EFA-def and Zn-def resulted in higher
22:5(n-6) levels (Table 4)
. For both liver PC and PE, Zn-def
reduced 20:3(n-6) levels (Tables 3
and 4
). For liver PC and PE, the
suckled control rats had higher DHA levels compared with the
artificially reared "physiologic control" rats. The overall
(n-6):(n-3) fatty acid ratio in liver phospholipids was lower in
suckled control rats compared with the artificially reared
"physiologic control" rats for both PC and PE liver phospholipids
(Tables 3
and 4
).
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The effects of the dietary manipulations were less pronounced in the
brain phospholipids compared with those of the liver and, in the brain,
were more clear for the PE than for the PC fraction. In brain PC,
EFA-def did not reduce AA, but there was a trend toward reduced DHA
levels (P = 0.09). EFA-def and Zn-def increased
22:5(n-6) levels in brain PC (Table 5
). In brain PE, EFA-def reduced both AA and DHA, and elevated
22:5(n-6) levels. Zn-def tended (P = 0.07) to
decrease AA levels, and Zn-def increased 22:5(n-6) levels only when
fed with EFA-adeq, as seen in the interaction between EFA and zinc.
In brain PE, Zn-def reduced 20:3(n-6) levels (Table 6
). In brain PE, the suckled control rats had lower AA and higher DHA
levels, whereas for PC, suckled control rats had higher AA and DHA
levels compared with the artificially reared "physiologic control"
rats. Only in brain PE was the (n-6):(n-3) fatty acid ratio lower in
suckled controls compared with the artificially reared "physiologic
control" rats (Tables 5
and 6
).
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The overall performance scores on the locomotor tasks are shown in
Figure 2
. Zn-def resulted in lower overall performance scores on all three
locomotor tasks (P < 0.001 for all tasks). No main
effect of EFA or an interaction between EFA and zinc was observed for
overall performance scores on the locomotor tasks.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) interacted with marginal dietary EFA deficiency to
influence liver and brain LCPUFA composition. As expected, EFA
deficiency reduced levels of AA and DHA in liver PC and brain PE. There
were significant interactions between zinc and EFA in liver such that
zinc deficiency reduced AA and DHA in the EFA-adequate groups and
increased AA (but not DHA) in the EFA-deficient groups. Thus,
contrary to the hypothesis, short-term zinc deficiency did not
exacerbate the effects of EFA deficiency. Both zinc and EFA deficiency
increased 22:5(n-6) levels in brain and liver phospholipids. An
increase in 22:5(n-6) has been proposed to be a compensatory mechanism
for a reduction in DHA (Galli et al. 1971
). In brain PE,
[which constitutes the greatest proportion of brain phospholipids
(Innis 1994
)], there was an interaction between zinc
and EFA such that zinc deficiency increased 22:5(n-6) in
EFA-adequate but not in EFA-deficient rats. In contrast,
although zinc deficiency also significantly reduced growth rates and
lowered the overall performance scores on locomotor skills tests, there
appeared to be no effect of EFA, nor an interaction between zinc and
EFA, on these outcome measures.
This is the first investigation to use the artificial rearing model to
address the interactive effects between EFA and zinc on liver and brain
LCPUFA composition and locomotor development in neonatal animals. Our
results are in contrast with earlier findings in mature animals. In
adolescent rats, zinc deficiency intensified the effects of EFA
deficiency on growth and skin lesions (Bettger et al. 1979
). In a more recent investigation in young adult rats, zinc
deficiency further reduced AA levels in liver PC and PE when rats were
fed a coconut oil–based diet that was low in EFA (Eder and Kirchgessner 1994a
). Interestingly, as was shown by the
interaction between zinc and EFA in this study, AA levels in liver
phospholipids were actually elevated by zinc deficiency in the
EFA-deficient groups. One explanation for this could be the fact
that desaturase activity appears to be regulated by the need for (n-6)
and (n-3) LCPUFA rather than by supply of LA and LNA (Innis 1994
). Thus zinc deficiency, in conjunction with EFA
deficiency, may have contributed to an up-regulation of desaturase
enzyme activity in rat pups because of their high need for LCPUFA.
However, because desaturase enzyme activity was not measured in this
study, this remains entirely speculative.
Zinc deficiency, independent of the EFA content of the diet, had only
small effects on the (n-3) and (n-6) LCPUFA in the PE and PC fractions
of the brain. It should be noted, however, that diet-induced
changes occur more slowly in the central nervous system than in other
tissues (Innis 1994
); in this study, the diet was fed
only for a period of 11 d. Further, the differences in brain zinc
concentrations between zinc-adequate and zinc-deficient rats
were much smaller compared with the differences in liver and plasma
zinc concentrations. This is consistent with previous suggestions of
the presence of considerable brain sparing with regard to zinc
homeostasis (Golub et al. 1995
, Prasad 1997
). Thus, even though brain zinc concentrations were
significantly reduced in zinc-deficient rats compared with
zinc-adequate rats, their brain zinc concentrations were actually
similar to those of the suckled control group. This may have
contributed to the small effects of zinc deficiency on brain fatty acid
composition.
The specific role that zinc plays in EFA metabolism is not clear. It
has been suggested that zinc is essential for 5- and 6-desaturase
enzyme activity, and a reduction of these enzyme activities has been
described in zinc-deficient animals (Clejan et al. 1982
). However, findings from this study and from others
(Eder and Kirchgessner 1994b
, Kudo et al. 1990
) do not support the suggestion that desaturase enzyme
activities were compromised to any great extent. In this study, lower
levels of 20:3(n-6) were observed in the zinc-deficient rats,
suggesting that 6-desaturase enzyme activity may have been
inhibited. However, findings of increased 22:5(n-6) levels and the
absence of higher LA levels in zinc-deficient rats do not support
this hypothesis. Caution should be exercised, however, in correlating
tissue LCPUFA levels with desaturase enzyme activity because of the
many other factors involved. Others have proposed that zinc deficiency
may affect either the affinity of acyl-CoA-lysophosphotidylcholine
acyltransferase for (n-3) and (n-6) fatty acids (Eder and Kirchgessner 1994a
), impair the regulation of desaturase
enzymes (Kudo et al. 1990
) or affect fatty acid
elongation, membrane lipid degradation and fatty acid oxidation
(Clejan et al. 1982
).
Only one other study has described the effects of zinc deficiency on
fatty acid composition of the brain during development (Yang and Cunnane 1994
). In that study, zinc deficiency clearly altered
the metabolism of (n-3) and (n-6) LCUFA and lowered total levels of
(n-3) and (n-6) LCPUFA in fetal brains of offspring from force-fed
zinc-deficient dams. However, that study did not present results of the
individual (n-3) and (n-6) LCPUFA, and also did not measure fetal brain
zinc concentrations. Furthermore, because the diet was fed to the
pregnant dams, it is not known whether the effects of zinc on fetal
brain fatty acid composition resulted from maternal or fetal zinc
deficiency.
Artificial rearing of rat pups resulted in a different brain and liver phospholipid composition with a higher overall (n-6):(n-3) fatty acid ratio of liver and brain phospholipids compared with suckled control rats. This was mainly a result of higher DHA concentrations in suckled control animals in liver and brain PE and PC compared with the physiologic control. This might be a result of the fact that the artificial rat formula did not contain any LCPUFA. It is also likely that other nonnutritional components in rat milk will affect fatty acid metabolism and incorporation of LCPUFA into tissues such as brain and liver.
Despite changes in brain phospholipid composition with EFA and zinc deficiency, as well as differences in fatty acid composition of brain phospholipids between artificially reared rat pups and suckled control rat pups, the behavioral outcomes as measured by overall performance on locomotor skill tasks were not affected by EFA deficiency but were clearly affected by zinc deficiency. The effects of zinc on locomotor skills were very pronounced, and because of the degree of peripheral zinc deficiency, were likely due to the apparent muscle weakness, and thus reduced capacity to ambulate. This interpretation is supported by the observation that brain zinc concentrations were similar or lower in the suckled control group compared with the artificially reared group. However, this group had the highest overall scores on these behavioral tasks. Thus, it is likely that no significant interactions between zinc and EFA were observed for the locomotor skills because of the strong effect of zinc, such that any effect of EFA, if present, could not be observed. Other tests batteries will be necessary to further characterize the behavioral consequences of zinc and EFA deficiency in early neonatal life.
Although the degree of zinc deficiency attained in this study was
more severe than that usually observed in a clinical setting, the
magnitude of the effects of the interaction with EFA on fatty acid
composition of brain and liver phospholipids was limited in its extent.
Nonetheless, these findings do support the conclusion that, when zinc
deficiency and EFA deficiency are present simultaneously, some
alterations in brain fatty acid metabolism can be anticipated. The
question is whether the small changes observed in fatty acid
composition in this study, although significant, are of any biological
importance. The small differences found in brain fatty acid composition
can be explained by the fact that the dietary manipulation was of short
duration and that, although the EFA-deficient diet was marginal, it
was not extremely deficient in (n-6) fatty acids. It is also possible
that the developing brain, in contrast to the mature brain, has sparing
mechanisms in place to ensure adequate incorporation of (n-3) and (n-6)
LCPUFA into brain phospholipids. It has been demonstrated, for example,
that the brain avidly retains (n-3) LCPUFA even during extended (n-3)
fatty acid deficiency (Futterman et al. 1971
,
Tinoco et al. 1977
and 1978
). Nonetheless, it is
probable that feeding such diets to artificially reared rats for a
longer period of time would have biologically significant effects. For
example, Wainwright et al. (1999)
have reported deficits
in working memory performance in 6-wk-old rats fed a similar marginally
EFA-deficient diet. It is therefore possible that the cumulative
effects of the interactions of dietary zinc and EFA deficiency may have
functional implications. This will require further investigation, but
using a less severe zinc deficiency in developing animals.
In summary, the results of this study suggest that short-term zinc deficiency does not exacerbate the effects of EFA deficiency on liver phospholipids and locomotor development in artificially reared rats, but does support the existence of a physiologic interaction between zinc and EFA status. Future studies should address the possible effects of long-term zinc deficiency and suboptimal EFA status on brain function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 I.P.W. is supported by a Post-Doctoral Fellowship Award from the National Institute of Nutrition, Canada.
4 Abbreviations used: AA, arachidonic acid; DHA,
docosahexaenoic acid; EFA, essential fatty acids; EFA-adeq,
essential fatty acid adequate; EFA-def, essential fatty acid
deficient; LA, linoleic acid; LCPUFA, long-chain polyunsaturated
fatty acids; LNA, linolenic acid; PC; phosphatidylcholine, PE;
phosphatidylethanolamine; Zn-adeq, zinc adequate; Zn-def, zinc
deficient.
Manuscript received March 5, 1999. Initial review completed April 22, 1999. Revision accepted June 23, 1999.
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