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,2
*
Department of Nutritional Sciences, University of California at Berkeley, CA 94720 and
Western Human Nutrition Research Center, USDA/ARS, San Francisco, CA 94129
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • rats • zinc • kinetics • pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A depressed rate of nucleic acid synthesis induced by
zinc deficiency may cause these anomalies (Hurley and Baly 1982
, Oteiza et al. 1988
). It is unlikely that
humans who are eating a diet composed of natural foods would consume a
diet virtually free of zinc; a low zinc intake is feasible, however, if
zinc-rich foods are not consumed. For that reason, experimental
studies of marginal zinc intakes are more applicable to humans. Studies
of the effects of marginal zinc intakes in pregnant rats and pregnant
rhesus monkeys have been done (Golub et al. 1984a and 1984b
, Herman et al. 1985
, Keen et al. 1989
). Unlike the studies using severely zinc-deficient
diets, marginal intakes of zinc prevented gross developmental defects
in both species. Litter sizes were normal, and placental function, as
measured by placental protein and DNA concentration and by the activity
of leucine aminopeptidase, a placental zinc metalloenzyme, was
unaffected.
The absence of any observable developmental defects suggests that
adjustments in zinc metabolism maintained embryogenesis when intakes
were marginal. After an intramuscular dose of
65Zn, Fairweather-Tait et al. (1985)
demonstrated that a greater portion of the dose was
transferred to the fetus and pups of dams fed a marginally
zinc-deficient diet compared with the pups of dams fed a
zinc-adequate diet (mean ± SEM; 0.084 ± 0.0057, 0.063 ± 0.0044, respectively). In addition,
65Zn uptake from an oral dose was lower in the
bones of marginally deficient pregnant dams than in the
zinc-sufficient group, suggesting that with inadequate zinc
intakes, pregnant rats mobilize zinc from metabolically active pools
for transfer to the fetus. Moreover, deposition of zinc into relatively
nonmobilizable maternal pools, i.e., bone, is reduced to preserve zinc
for essential functions.
Plasma zinc is a major component of the metabolically active zinc pool.
In a previous study of nonpregnant rats, we developed a simple model of
zinc metabolism describing short-term zinc kinetics (Lowe et al. 1991
). In this model, the disappearance of an isotopic
tracer of zinc (65Zn) over 120 min is described
by two compartments, pool a and pool b. The size, turnover rates and
flux of zinc from these two pools are altered by acute zinc depletion
and endotoxin infection (Lowe et al. 1991
). We
hypothesize that the kinetics of zinc in these two compartments are
altered when the diet provides marginal amounts of zinc during
pregnancy, particularly during the last 4 d of gestation when the
demand for zinc is high due to rapid fetal growth (Hurley and Baly 1982
). To test this hypothesis, we measured short-term
zinc kinetics in two groups of rats; one group was fed an adequate
amount of zinc (30 µg/g), and the second group received a
marginal zinc intake (6 µg/g). Half of the animals in each
group were mated after consuming these diets for 6 wk. Because severe
zinc deficiency causes a reduction in food intake (Giugliano and Millward 1984
), a pair-fed group was also studied during
pregnancy. All rats were killed after 9 wk or on the last day of
gestation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A third group of pair-fed rats was also studied (n = 6). This group was given free access to the control diet from weaning until 6 wk of age. After mating, each pregnant dam (PFPG) was paired with a pregnant dam from the 6 Zn group (6ZnPG). The weight of food given to each PFPG dam was equal to that consumed by her partner in the 6ZnPG group on the same day of gestation.
Isotope studies.
On d 20 of pregnancy or after 9 wk of consuming the experimental diet for the nonpregnant groups, the rats were weighed and anesthetized with a single bolus dose of sodium pentobarbital (5 mg/100 g rat weight). A cannula (size 2FG, Portex, Kent, England) was inserted into the carotid artery and a baseline blood sample (0.4 mL) was taken. A syringe containing 92.5 MBq 65Zn in 0.5 mL sterile isotonic saline was weighed and the isotope injected into the rat via the femoral vein. The empty syringe was weighed and the weight of the isotope administered was recorded. Blood samples (0.4 mL) were taken via the cannula at 2.5, 5, 10, 15, 30, 45, 60, 75, 90 and 105 min post-65Zn injection and placed on ice. The cannula was flushed with heparinized saline after each blood draw to prevent clots from forming. Plasma was immediately separated from whole blood by centrifugation at 13,600 x g for 3 min (Micro-Centrifuge Model 235C, Fisher Scientific, Pittsburgh, PA). An aliquot of 0.2 mL plasma was removed with the use of a positive displacement pipette, placed in an acid-washed polyethylene tube and stored on ice.
After the final blood sample was drawn, the rat was exsanguinated. The intact uterus was removed, weighed and the number of fetuses recorded. Each fetus was weighed and examined for gross abnormalities. The fetus from the top of the right uterine horn was saved for zinc analysis. The maternal lungs, liver, spleen, kidneys and femurs were also removed. All tissues were blotted to remove any excess blood, then weighed and stored at -20°C. All plasma samples were counted on a gamma counter (Searle Model 1197 Automatic Gamma system, Searle Analytic, Des Plaines, IL) at the end of the procedure.
Tissue analysis.
All soft tissues (whole liver, whole spleen, both lungs and both kidneys) were homogenized in 10 mL of distilled water by using a polytron fitted with a stainless steel probe (PT 21, Kinematica GmbH, Lucerne, Switzerland). A sample of each homogenate (2 g) was weighed into a polystyrene tube (Sarstedt 55.461, Hayward, CA) and counted in a gamma counter. Femur samples were dissolved in 1 mL concentrated HNO3 overnight before counting in a gamma counter. The number of counts per minute per whole tissue sample was calculated and expressed as a percentage of the dose given.
Total zinc determination.
Soft tissues. A sample of tissue homogenate (0.5 g) was weighed into an acid-washed, glass dish and gently heated on a hot plate until dry. Homogenates were then ashed overnight in a low temperature asher (Model LTA-604, International Plasma, Hayward CA) and dissolved in 25 mL of 0.125 mol/L HNO3. The zinc concentration of the dissolved ash was determined by atomic absorption spectrophotometry (AAS) (Smith-Hieftje 22, Thermo Jarrel Ash, Franklin, MA)
Bone. The dissolved femur samples were diluted volumetrically in 200 mL of distilled water. To bring the zinc concentration within the standard range if necessary, the sample was further diluted with 0.125 mol/L HNO3 before measuring the zinc concentration by AAS.
Plasma. One milliliter of 0.125 mol/L HCl was added to each 0.2-mL plasma sample and mixed thoroughly. The concentration of zinc was then determined by AAS. The specific activity of the plasma at each time point was calculated by dividing the number of counts per second by the total amount of zinc in each sample.
Kinetic analysis.
A curve of the decay of plasma specific activity vs. time was analyzed
by using a computerized program for regression analysis (Blackwell
Scientific Software, Oxford, UK). The minimum number of exponential
terms required to describe each plasma decay curve was determined. The
data were fit to a compartmental model (Lowe et al. 1991
, Shipley and Clark 1972
).
Statistics.
Statistical analyses were done using Microsoft Excel, version 5.0 (Microsoft Corporation, Redmond, WA). Comparisons between the 30Zn and 30ZnPG groups and between the 6Zn and 6ZnPG groups were made using a two-tailed, unpaired Student's t test. Comparisons among the three groups of pregnant rats (30ZnPG, 6ZnPG and PFPG) were made using one-way ANOVA and Tukey's post-hoc test. Significance was set a priori at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the first 6 wk after weaning, the 6Zn rats gained less weight
than the 30Zn rats; the difference was significant after 1 wk
(Fig. 1
). At the time of mating, 6 wk post-weaning, the 6Zn group weighed
(±SD)181.2 ± 9.7 g, whereas the 30Zn group
weighed 190.3 ± 11.7 g. The 6ZnPG and PFPG groups gained
less weight during pregnancy (Fig. 2
) than the 30ZnPG group (Table 1). Food consumption of the 6ZnPG group was generally less than that
in the ZnPG group and declined in late gestation, falling from
16.2 g on d 17 to 3.8 g on d 19 (Fig. 3
).
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Pregnancy outcome did not differ among the three groups. The number of
fetuses, mean fetal weight and total weight of the conceptus did not
differ (Table 1)
. There were no gross congenital
anomalies.
Tissue zinc concentration and distribution of the 65Zn dose.
In the 30Zn groups, pregnancy did not alter tissue zinc concentrations
(Table 2). The percentage of the 65Zn dose retained in the
lung, kidney and femur 105 min after isotope administration, however,
was significantly lower in the pregnant dams compared with the
nonpregnant controls (Table 3). In the 6Zn groups, the concentrations of zinc in the plasma and
kidneys were 65 and 6% lower, respectively, in the pregnant dams
compared with the nonpregnant rats (Table 2)
, but the retention of the
65Zn dose in the tissues did not differ (Table 3)
.
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. Femur zinc
concentrations were significantly lower in the 6ZnPG group compared
with the other two groups, and in the PFPG group compared with the
30ZnPG group. The kidney zinc concentration in the 6ZnPG group was
lower than that of the PFPG and 30ZnPG rats (Table 2)
. A higher
percentage of the 65Zn dose was retained in the lung of the
6ZnPG rats compared with the 30ZnPG and PFPG controls, and there was a
higher percentage of the dose present in the fetuses of the 6ZnPG dams
compared with those of the 30ZnPG dams (Table 3)
. Kinetic studies.
Because the equation for the plasma specific activity decay curve for
the pregnant and nonpregnant rats had two exponential terms, a
two-compartment model was used to describe the zinc kinetics for
the study (Shipley and Clark 1972
). The exponential
equation and the dose of 65Zn administered were used to
calculate the size of pool a (Qa), and pool b (Qb), the fractional
turnover rates of the pools (k values) and the flux of zinc between the
pools (F values) (Fig. 4
) (Lowe et al. 1991
). A typical fit of the plasma
specific activity to the model for each group of rats is shown in
Figure 5.
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). However, in the 6ZnPG group, the sizes of pools a and b were 54
and 35% lower, respectively, than those of the 6Zn group. The 6ZnPG
rats also had an elevated fractional turnover rate (kaa) and an
increase in the fraction of pool a moving to pool b (kba) and to other
tissues (koa) (P < 0.02). The flux, or amount of
zinc, moving between pools a and b (Fab) was 40% lower
(P < 0.02) in the 6ZnPG than in the 6Zn group
(Table 4)
.
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. This was
associated with a higher fractional turnover rate of pool a to sites
other than pool b (koa) and a lower zinc flux between the two pools
(Fab). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) show that the supply of
zinc to the placenta is sufficient to support fetal growth in chronic
marginal zinc deficiency. The zinc kinetics demonstrated that a chronic
marginal zinc intake during pregnancy reduced the size of two pools of
zinc that exchange with the isotope in a 2-h period. Previous work has
demonstrated that pool a is composed of plasma zinc and pool b is
composed of a portion of the zinc present in the liver (Lowe et al. 1991
). The reduction in Qb observed in the 6ZnPG group
(Table 4)
compared with the 30ZnPG and PFPG groups was not accompanied
by a fall in the liver zinc concentration (Table 2)
. The average weight
of the liver in the 6ZnPG group was significantly lower than that of
the 30ZnPG group (66 vs. 87 mg, respectively). However, the
pair-fed group also had small livers (60 mg) but no reduction in
Qb. The likely explanation for this is that Qb is only a small portion
of the total liver pool and is selectively mobilized in conditions of
dietary zinc deficiency. Previous work has shown that Qb constitutes
only 6% in Zn-depleted animals and up to 40% in
zinc-sufficient animals (Lowe et al 1991
).
This reduction in exchangeable zinc was associated with an increase in
the fractional turnover rate of pool a and fraction of zinc moving from
pool a to other tissues. Although the fractional turnover of zinc from
pool a increased, the amount of zinc moving from pool a to pool b was
reduced as a result of the reduction in the size of pool a. There was
no significant decrease, however, in the amount of zinc moving out of
pool a to the other tissues. Thus, despite the fall in the size of pool
a, zinc flux out of pool a to other tissues (Qa x koa), was
sustained close to control values. This could be achieved in part by an
increase in the proportion of the circulating plasma zinc that is bound
to albumin, which is more labile than that bound to
-2-macroglobulin. Studies in humans have shown that the proportion
of zinc bound to albumin is increased in pregnant women who are
marginally deficient in zinc (Fehily et al. 1987
).
The concentration of zinc in maternal tissues was not affected by the
state of pregnancy when the dietary supply of zinc was adequate. When a
diet with marginal amounts of zinc was fed, only the plasma and kidney
zinc concentrations were lower in pregnant compared with nonpregnant
animals. Others have shown that maternal tissue zinc concentrations are
unchanged, even in conditions of severe, teratogenic zinc deficiency
(Hurley and Swenerton 1971
). Pregnant dams are unable to
mobilize tissue zinc from maternal tissues to support fetal growth and
development unless tissue catabolism is induced by a marked reduction
in food intake (Apgar 1975
, Masters et al. 1983
). Pregnant rats fed a severely zinc deficient diet (0.75
µg/g) had a marked fall in voluntary food intake on d 18
of gestation, which caused tissue catabolism and the release of
substantial amounts of zinc when fetal needs are the highest
(Masters et al. 1983
). In this study of marginal zinc
intakes, the food intake of the dam also declined dramatically on d 18
of gestation, and tissue catabolism occurred, leading to a reduction in
body weight (Figs. 2
and 3)
. Food intake did not fall in the control
animals. This mobilization of maternal tissue zinc in the marginal zinc
group along with the increase in turnover rate of the plasma zinc pool
enabled the dams to deliver apparently normal, healthy pups.
When dietary zinc is sufficient, pregnancy had no effect on the size,
turnover rate and flux of zinc between the two rapidly exchanging
metabolic pools. However, the retention of the
65Zn dose 105 min after administration was lower
in bone, lung and kidney of the pregnant dams fed adequate amounts of
zinc than in the nonpregnant dams. This supports the hypothesis that
zinc uptake by tissues that turn over more slowly is reduced in late
pregnancy (Fairweather-Tait et al. 1985
).
In summary, the results of this study show that when dietary zinc is marginal, the flux of zinc to the rapidly growing fetus is sustained by a reduction in the zinc flux to maternal hepatic pools and to other more slowly turning over tissues, such as bone. These adjustments in zinc kinetics, along with a reduction in maternal food intake and tissue catabolism in late gestation, supported a normal pregnancy outcome. Further studies are required to determine the mechanisms by which these maternal adjustments in zinc kinetics occur.
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FOOTNOTES |
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3 Abbreviations used: AAS, atomic absorption
spectrophotometry; Fao, rate of zinc flux from outside the system into
pool a; Fab, rate of zinc flux from pool a to pool b; kaa, fractional
turnover rate of pool a; kbb, fractional turnover rate of pool b; kba,
fraction of pool a moving to pool b; koa, fraction of pool a moving to
outside the system; Qa, size of pool a; Qb, size of pool b; PFPG,
pair-fed pregnant rats; 6Zn, nonpregnant rats fed a diet containing
6 mg Zn/g diet; 30Zn, nonpregnant rats fed a diet containing 30 mg Zn/g
diet; 6ZnPG, pregnant rats fed a diet containing 6 mg Zn/g diet;
30ZnPG, pregnant rats fed a diet containing 30 mg Zn/g diet.
4 The control diet contained the following (g/kg):
egg whites (spray-dried), 200; cornstarch, 150; sucrose, 496.996;
cellulose, 50; corn oil, 50; salt mix (zinc-deficient, AIN-76), 35;
vitamin mix (AIN-76A), 10; biotin, 0.004; choline bitartrate, 2;
zinc-sucrose premix (5 mg Zn/ g), 6. The Zn-deficient diet
contained sucrose alone rather than the zinc-sucrose premix.
Manuscript received September 10, 1998. Initial review completed October 7, 1998. Revision accepted January 27, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1. American Institute of Nutrition Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 1977;107:1340-1348
2. American Institute of Nutrition Second report of the ad hoc committee on standards for nutritional studies. J. Nutr. 1980;110:1726
3. Apgar J. Zinc and reproduction. Annu. Rev. Nutr. 1985;5:43-68[Medline]
4. Fairweather-Tait S. J., Wright A.J.A., Cooke J., Franklin J. Studies of zinc metabolism in pregnant and lactating rats. Br. J. Nutr. 1985;54:401-413[Medline]
5. Fehily D., Kiswani A. S., Jenkins D. M., Cremin F. M. Redistribution of plasma zinc between albumin and alpha 2-macroglobulin in pregnancy. J. Trace Elem. Electrolytes Health Dis. 1987;1:83-88
6. Giugliano R., Millward D. J. Growth and zinc homeostasis in the severely Zn deficient rat. Br. J. Nutr. 1984;52:545-560[Medline]
7.
Golub M. S., Gershwin M. E., Hurley L. S., Baly D. L., Hendrickx A. G. Studies of marginal zinc deprivation in rhesus monkeys. I. Influence on pregnant dams. Am. J. Clin. Nutr. 1984;39:265-280
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Golub M. S., Gershwin M. E., Hurley L. S., Baly D. L., Hendrickx A. G. Studies of marginal zinc deprivation in rhesus monkeys. II. Pregnancy outcome. Am. J. Clin. Nutr. 1984;39:879-887
9. Herman Z., Greeley S., King J. C. Placenta and maternal effects of marginal zinc deficiency during gestation in rats. Nutr. Res. 1985;5:211-219
10. Hurley L. S. Maternal catabolism and fetal zinc status. Nutr. Res. 1985;(suppl I):300-305
11. Hurley L. S., Baly D. L. The effects of zinc deficiency during pregnancy. Prasad A. S. eds. Clinical, Biochemical and Nutritional Aspects of Trace Elements 1982:149-159 Alan R. Liss New York, NY.
12. Hurley L. S., Swenerton H. Lack of mobilization of bone and liver zinc under teratogenic conditions of zinc deficiency in rats. J. Nutr. 1971;101:597-604
13. Keen C. L., Lonnerdal B., Golub M., Uriu-Hare J. Y., Olin K. L., Hendrickx A. G., Gershwin M. E. Influence of marginal maternal zinc deficiency on pregnancy outcome and infant zinc status in rhesus monkeys. Pediatr. Res. 1989;26:470-477[Medline]
14. Lowe N. M., Bremner I., Jackson M. J. Plasma 65Zn kinetics in the rat. Br. J. Nutr. 1991;65:445-455[Medline]
15. Masters D. G., Keen C. L., Lonnerdal B., Hurley L. S. Zinc deficiency teratogenicity: the protective role of maternal tissue catabolism. J. Nutr. 1983;113:905-912
16. Oteiza P. I., Hurley L. S., Lonnerdal B., Keen C. L. Marginal zinc deficiency affects maternal brain microtubule assembly in rats. J. Nutr. 1988;118:735-738
17. Shipley R. A., Clark R. E. Tracer Methods for "in vivo" Kinetics 1972 Academic Press New York, NY.
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