BIOLOGICAL TRACE ELEMENT RESEARCH 4, 221-232 (1982)
Consecutive Zinc Balance Trials in Growing Rats H. G.
PETERING,*
E.
GIROUX,~
H.
CHOUDHURY,
AND E . E . M E N D E N
Institute of Environmental Health, Kettering Laboratory, University of Cincinnati Medical Center, Cincinatti, Ohio 45267 and
Merrell Research Center, Merrell Dow Pharmaceuticals, Cincinnatti, Ohio 45215 Received March 25, 1982; Accepted April 9, 1982
Abstract Rats were fed a purified egg white-based diet containing 5 ppm Cu and 2, 14, or 57 ppm Zn. Zinc and copper balances were determined for eight consecutive weekly trial periods. The zinc-deficient group almost ceased to gain weight and was in slightly negative zinc balance. Groups of rats fed 14 and 57 ppm Zn gained weight at equal rates. These groups were in strongly positive zinc balance for four weeks; thereafter, they were in slightly positive zinc balance. Over the eight week series of balances, the group fed 57 ppm Zn retained about two times as much zinc as did the group fed the diet containing 14 ppm Zn. All groups were in null or slightly negative copper balance throughout the trial. These results suggest that zinc accumulation may be homeostatically controlled to a level in excess of that needed for maximum growth. Index Entries: Zinc balance; homeostasis, of zinc; copper balance; optimal nutriture; growth rate effect, of Zn; effect of zinc on copper metabolism.
Introduction A series of reports by Weigand and Kirchgessner (1-8) has defined and analyzed factors that describe zinc utilization by the laboratory rat. Apparent retention is de9 1982 by The Hurnana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/82/6900-0221 $02.40
221
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PETERING ET AL.
termined by analyses of total zinc in balance studies and is the difference between intake and excretion. The latter factor is composed of unabsorbed dietary zinc (fecal excretion only), inevitable metabolic losses, and excretion to maintain homeostasis (5). True absorption of zinc and the factoring of zinc excretion into its several components is assessed by radioisotopic techniques (1, 2). Balance studies typically are carried out for trial periods of 1 week, following an interval for adaptation of animals to the test diet. In the experiments reported below, we measured copper and zinc intake and excretion during eight consecutive weekly trial periods. Zinc balance varied considerably over the course of the study. One principal conclusion we reached was that rapidly growing rats may retain zinc in excess of that needed for maximum weight gain and that the accumulation of zinc slows as the rate of weight gain lessens. Thus, nutritional adequacy may be achieved at a level of dietary zinc below that which activates homeostatic mechanisms. Generalizations about zinc metabolism measured in short-term trials may be highly dependent on the age, dietary history of the test animals, and on other experimental conditions.
Methods Three purified diets that differed only in their contents of zinc acetate were prepared with the composition indicated in Table 1. They met NRC recommendations for all ingredients, except for the dietary levels of 2, 14, and 57 ppm Zn that were chosen to provide deficient, marginally sufficient, and abundant zinc nutriture, reTABLE 1 Composition of Diet" Diet component
g/kg
Egg white (spray-dried) Cornstarch Glucose Corn oil Mineral mixb Cellulose powder Oil-soluble vitamins C Water-soluble vitamins " Choline chloride
200.0 433.5 200.0 75.0 40.0 30.0 15.0 5.0 1.5
aDiet, except for vitamin and mineral additions, was prepared by Zeigler Bros., Inc., Gardners, PA. ~ Bernhart and Tomarelli (15) salt mix supplied (per kg diet): cupric sulfate(5 mg Cu), chromic acetate (2 mg Cr), ammoniummolybdate(0.5 mg Mo), sodium selenite (0.1 mg Se), and zinc acetate (2, 14, or 57 mg Zn). cPetering et al. (16).
ZINC BALANCE IN RATS
223
spectively. Sprague-Dawley male weanling rats (3 weeks old) with an initial weight range of 37-58 g were obtained from Charles River (Portgage, MI) and were housed individually in stainless steel cages under conditions designed to minimize their inadvertent exposure to metals (9). All rats were fed the diet containing 14 ppm Zn for an adaptation period of 3 weeks. At the age of 6 weeks they were allocated to three groups of 10 animals so that the mean body weight of each group was 171 g. One group was fed a diet containing 57 ppm Zn (by analysis) and one group was continued on diet containing 14 ppm Zn (by analysis). These diets were fed for 8 weeks. One group was fed a diet containing 1.1 ppm Zn for 2 weeks, then a diet containing 2.4 ppm Zn for 6 weeks; the intention had been to feed a diet of ca. 2 ppm Zn to the latter group for the entire period of 8 weeks, but metal analyses revealed an error, as indicated, in the preparation of the first batch of zinc-deficient diet. For every rat, weekly water and food intakes were recorded, as was body weight; feces were collected each day and pooled for each rat on a weekly basis. On one day in each of the 8 weeks of the experimental period, five rats from each of the three groups were fasted for 24 h, with free access to deionized water being permitted; during this time a complete collection of urine was made. Each rat was subjected every other week to this protocol. We assumed that data obtained from half of each group were representative of the whole group. This protocol was imposed in order to prevent dietary contamination of urine collections. After eight weeks of feeding the three diets, the rats were bled and subsequently killed and necropsied. Weights of liver, heart, pancreas, kidneys, and testes were recorded. Portions of these organs, plus serum, different batches of diet, and representative samples of excreta were submitted to analysis of their zinc and copper contents. Metal analysis was by atomic absorption spectroscopy (Perkin-Elmer Model 403); sample preparation and quality control for such analyses has been described (10).
Results Growth, Feed Consumption, and Feed Efficiency Growth curves for the groups of rats fed the three diets over the 3-week conditioning period and the 8 weeks of the experimental balance period are illustrated in Fig. 1. Rats fed the diet containing 14 ppm Zn maintained a constant rate of increase in body weight of 49 -+ 1 g (SEM) per week up to the end of the seventh week (fourth week of balances) when the mean weight was 367 + 9 (SEM). During the last 4 weeks of the study the weight gain was 25 -+ 4 g/week. There was no effect on weight gain for the group of rats shifted from the diet containing 14 ppm Zn to the one containing 57 ppm Zn. During the first 4 weeks on this diet these animals gained 49 -+ 3 g/week, while during the last 4 weeks they gained 22 -+ 3 g/week. Rats shifted to the Zn-deficient diet almost ceased to gain weight. During the last 4 weeks of the study this group of rats maintained a mean body weight of
224
PETERING
ET AL.
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Fig. 1. Growthcurves for groups of 10 rats fed for three weeks with egg white-based diet containing 14 ppm Zn, then shifted to zinc-deficientdiet or zinc-abundantdiet, or continued on diet containing 14 ppm Zn. Vertical bars denote SEM. 197 -+ 1 g. The total mean increase of body weight of this group for the 8 weeks of the experimental period was 27 g compared to total increases of 295 and 283 g for the groups fed the 14 and the 57 ppm Zn diets, respectively. Our data for feed consumption and its efficiency were consistent with the observations of Weigand and Kirchgessner (3) that food intake of rats may be precisely defined by a function of live weight and weight gain. The groups fed diets containing 14 and 57 ppm Zn had comparable feed intakes as well as comparable body weight (BW) gains during each weekly interval. On the other hand, the feed consumption was substantially less for the group receiving the Zn-deficient diet. During the first 2 weeks the Zn-deficient group ate 39 and 41 g/100 g BW, respectively, while the other two groups consumed an average of 56 and 52 g/100 g BW for these two weekly periods. Feed consumption relative to body weight during the last 6 weeks was comparable in all groups and declined from an average of 44 g/100 g BW for the third week to an average of 35 g/100 g BW the last week. Over the first 4 weeks of the balance period, the Zn deficient group gained 10.7 g/100 g of feed consumed, while the two other groups gained more than three times this amount, namely 35 g/100 g of feed consumed. Feed efficiency values fell considerably over the last four weeks of the balance study. The Zn-deficient group had
ZINC BALANCE IN RATS
225
an efficiency value of - 1.6 g/100 g feed, while the adequate and excessive dietary zinc groups had efficiencies of 17.4 and 15.0 g/100 g of feed consumed, respectively.
Zinc and Copper lntake, Excretion, and Retention Mean values per rat per week of zinc and copper intake, excretion, and retention for each group summed over the first and second 4-week halves of the experimental period (w4-wl 1) are given in Table 2. The weekly values for intake and excretion relative to body weight for the group consuming the 14 ppm Zn diet are presented in Fig. 2 as illustrative of the time relationships in this group and in the one receiving the diet with the highest amount of zinc. In addition, the weekly retention values for each group in relation to body weight are presented in Fig. 3. The intakes of zinc and copper are obviously related to the feed consumption of each group as well as to the dietary metal content. Thus, during the 8-week balance period (w4-wl 1) the rats fed the diet containing 14 ppm of Zn ingested about 14 times the amount of Zn ingested by the rats in the Zn-deficient group. Furthermore, the group receiving 57 ppm Zn in the diet ingested 4 times the amount of the group receiving 14 ppm dietary Zn. There was, therefore, a fiftyfold range in the weekly intake of Zn among the three groups of rats. Over the 8-week experimental period copper intake of the Zn-deficient group amounted to 2.8 mg per rat, while the total intake of copper amounted to 5.7 mg per rat in the two other groups, i.e., double the amount of the Zn-deficient group. This was to be expected, since dietary levels were 5 ppm Cu in each diet, and feed consumption of the Zn-deficient group was one-half that of the two other groups. The amount of zinc or copper excreted in urine was very small in every group and never exceeded 0.2 mg Zn or 0.02 mg Cu per rat per week, even in the group receiving 57 ppm Zn in the diet. Nevertheless, urinary Zn did account for almost one-third of the total excretory value for the Zn-deficient group, which was in contrast to 5 and 1% of the total excretion values for the 14 and the 57 ppm dietary Zn groups, respectively. At each level of dietary Zn, fecal excretion of either Zn or Cu TABLE 2 Zinc and Copper Balance Group mean, a mg/rat Zn-deficient, 2 ppm Zn-adequate, 14 ppm Period
A. Zinc w4-w7 w8-wll B. Copper w4-w7 w8-wll
Zn-excessive, 57 ppm
I
E
R
I
E
R
I
E
R
0.53 0.64
0.73 0.99
-0.20 -0.35
8.02 8.12
2.97 5.71
+5.06 +2.41
32.15 32.19
20.35 30.31
+11.81 + 1.88
1.45 1.33
1.33 1.28
+0.12 +0.05
2.81 2.84
2.63 3.58
+0.18 -0.74
2.82 2.90
2.48 3.33
+ 0.34 - 0.43
aI zinc or copper intake for each 4 week period; E = the sum of weeklyfecal and urinary excretion for each 4 week period; R = I - E and is the overall retention for the sum of data from each of 4 weeks. =
226
PETERING ET AL.
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Fig. 2. Weekly consumption of dietary zinc (filled circles), fecal zinc excretion (open circles), and urinary zinc excretion (filled squares), for 10 rats fed a diet containing 14 ppm Zn. Group mean values are expressed per kg body weight; over 8 weeks the animals grew from a mean weight of 171 to 466 g. Vertical bars denote SEM. was the major route of excretion of these metals. There was no consistent pattern of variation with time for the fecal Zn excretion of the rats in the Zn-deficient group, but there was a progressive increase in fecal zinc in the two other groups. The same pattern was found for copper fecal excretion, namely, no consistent time variation for the Zn-deficient dietary group, but a consistent increase in weekly fecal copper values for the other two groups. The group of rats receiving the Zn-deficient diet was consistently in slightly negative Zn balance, which was somewhat greater in the second half of the balance period than in the first. Rats fed 14 ppm dietary zinc retained 63% of the zinc they ingested in the first 4 weeks of trial periods and 30% of ingested zinc in the last 4 weeks. Rats fed 57 ppm dietary zinc retained 39 and 6% of ingested zinc in the same two intervals, respectively. The 57 ppm Zn group retained more than twice as much zinc during the first half of the balance trials as did the group consuming the 14 ppm Zn diet. The 57 ppm Zn group retained slightly less than twice as much Zn as the 14 ppm Zn group for the entire 8-week period. The highest weekly retention in proportion to BW for both the 14 and 57 ppm Zn groups occurred in the first week. Thereafter, the retention values declined until the fifth week of balances, after which relatively stable positive values were recorded. It was at this point, namely, week 8, that these two groups changed their growth patterns. Over the 8-week balance period the group fed the diet with the highest content of zinc retained 21% of its intake, while the group fed the 14 ppm Zn diet retained 47% of its
ZINC BALANCE IN RATS
227
0 57 ppm Zn 9 14ppm Zn
16
u
2 ppm Zn
#,
8
0 I 4
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Fig. 3. Weekly zinc balances for groups of 10 rats fed egg white-based diets containing zinc concentrations characterized as deficient, adequate, or abundant. Zinc retention is expressed per kg body weight; the zinc-deficient group increased from a mean body weight of 171 to 197 g over the 8-week period, whereas the two other groups grew to about 460 g. intake. Thus, these two groups, which did not differ substantially in either growth rate or feed consumption, did differ markedly in their retention of Zn. Copper retention in the group consuming the Zn-deficient diet varied weekly between slightly negative and slightly positive values, but the mean absolute retention values per rat in both halves of the experiment were slightly positive. In the cases of the two other groups, the copper excreted over the 8 week period was 115% and 102% of that consumed for the 14 and 57 ppm Zn groups, respectively. In both groups, retention was slightly positive in the first half and slightly negative in the second half of the experiment.
Tissue Zinc and Copper Concentrations Tissue metal data presented in Table 3 were obtained at the end of the experiment. The concentrations of zinc in kidney, pancreas, serum, and testes of the group receiving the Zn-deficient diet were statistically lower than those of the two other groups. There was a statistically significant difference between the kidney concentrations of zinc in the two groups receiving the higher dietary levels of Zn. A dose-
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response relationship appeared to hold between Zn intake and concentration in kidney, liver, and pancreas. The only effect that varying dietary zinc had on tissue copper concentrations was evident in testes, where it was found that the copper concentration of the testes of the group receiving 2 ppm dietary Zn was much higher than the values for the two other groups. The specific weights of the organs examined, i.e., the weight of the organ relative to body weight, were quite similar, as might be expected.
Discussion This experiment presents a study of the effects of feeding diets varying twentyfivefold in Zn concentration on zinc and copper balance in growing rats over a longer period of time than has usually been studied. These balances were observed during a transition from a younger physiologic state to a more mature one, as is shown by the changes that occurred in growth rates and feed intakes of the rats after the tenth week of life. Because the trials were conducted over an 8-week period, we had no reliable and useful method for continuous evaluation of true absorption and intestinal excretion of either zinc or copper. Our data, therefore, relate to balance patterns under our experimental conditions where fecal values include unabsorbed dietary zinc and copper, together with intestinally excreted zinc and copper. If weight gain is used as a criterion of nutritional adequacy, our test diets containing 14 and 57 ppm Zn would be judged equivalent. This conclusion is in line with results of Forbes and Yohe (11), who showed that 5-week weight gain of young rats is maximized when casein- or egg white-based diets contain 12 ppm Zn or greater amounts. Furthermore, the National Research Council has established (12) that a purified casein-based diet containing 12 ppm Zn meets known nutritional requirements of the rat. We labeled as abundant zinc the diet containing 57 ppm Zn. This was simply a relative designation as this level of zinc is about twice the 30 ppm level recommended for purified diets by the American Institute of Nutrition (13), but as the data show, it was four times that required under our conditions for optimal growth. An important concept in the model of homeostatic regulation of zinc metabolism is that the organism does not retain metal in excess of an optimum level that contributes to body maintenance and productive function at the time of its deposition (8). Thus, our observation that rats fed the diet containing 57 ppm Zn accumulated two times as much zinc over the 8 weeks of balance trials as did rats fed the diet containing 14 ppm Zn, implies that the latter diet was not optimal for all needs. Weigand and Kirchgessner (6), using a casein-based diet fed to weanling rats, showed in 6 day balance trials that apparent retention of zinc reached a plateau when dietary zinc concentration was 39 ppm or greater; at 10.6 ppm, apparent retention of zinc was less than half the plateau level. Our results are consistent with theirs on this point. However, Weigand and Kirchgessner found that the group fed a diet containing 10.6 ppm Zn had a mean growth rate 77% of that of the group fed at the higher level (8). The maximum weight gain of their rats was 6.2 g/day,
230
P E T E R I N G ET A L .
slightly less than the gain of 7 g/day we observed in the groups of rats fed diets containing 14 and 57 ppm Zn. The rats achieving homeostasis in their trials were in strongly positive zinc balance (5). Elsewhere (7), these authors calculated that rats of 200 g body weight would display an irreducible fecal elimination of endogenous zinc of 11-13 ~g/day. During the last 4 weeks of our study, the group of zincdeficient rats exhibited a mean negative balance of 12.5 p~g Zn/day. In principle, tissue metal concentrations indicate homeostatic balance as well, and might be employed to complement retention data. The inadequacy of the zincdeficient diet may be illustrated on this basis, since tissue zinc concentrations in rats fed this diet were significantly below those recorded in rats fed the diet containing 57 ppm Zn. The extra zinc retained by rats fed the zinc-abundant diet, compared to those fed 14 ppm dietary zinc, may be calculated to have raised wholebody zinc concentration by 6 mg/kg; and although tissue zinc concentrations that we determined were almost uniformly lower in the group fed at the lower level of dietary zinc, analytical imprecision limited us in assigning statistical significance to the differences. Weigand and Kirchgessner (4) also reported that deposition of zinc per gram of live weight gain is reflected better in whole body zinc than in zinc concentrations in liver, kidney, pancreas, and small intestine. A large amount of body zinc is found in bones, as indicated by the report of E1-Gazzar et al. (14). An inference that can be drawn from these observations is that a diet adequate for growth may not be an optimal one, if optimal is defined by activation of homeostatic regulatory mechanisms. By this definition, maximum growth rate may be insufficient as an indicator of optimal nutriture. It is unclear what functions beyond maximum growth require the increment in zinc that is the difference between adequate and optimal levels. Whatever these functions may be, they appear to be transitory: halfway through the 8 weeks of balance trials, i.e., at 10 weeks of age, the group of rats fed at the 57 ppm level of dietary zinc went into almost null balance with respect to this metal. Interestingly, the group of rats fed the 14 ppm Zn diet containing an adequate amount of zinc for growth went into a similar balance with respect to zinc at this point. Prior to this time, the latter group accumulated only half the zinc accumulated by the former group. One interpretation of this observation is that retention is homeostatically regulated to provide an excess of zinc over that necessary for all immediate metabolic requirements. This possibility is considered in the model of Weigand and Kirchgessner, but is hypothesized to occur only in special situations such as during pregnancy or disturbed metal utilization (5). In their model there is no inconsistency between adequate and optimal nutriture: dietary intake that just permits homeostatic regulation to operate is considered the basal or minimal gross requirement. Further study may clarify whether superretention of zinc generally occurs. Our sequential collection of balance data illustrates that single trials may not yield enough information for understanding all of the nutritional need for Zn. In particular, we have the opinion that the shift from a very positive zinc balance, which appears more a function of body weight (or age) than a function of zinc accumulation, may provide a lead in furthering our understanding of zinc metabolism.
ZINC BALANCE IN RATS
231
The metabolism of zinc and copper have been linked in many ways by many investigators, so we determined copper balance during the entire experiment in all three groups of rats; despite the more than twentyfold variation in dietary Zn/Cu ratio, we did not find outstanding differences in copper metabolism. Copper balance was slightly negative in both 14 and 57 ppm Zn groups of rats. Since those groups grew from mean weights of 170 to 460 g during the trials, the whole-body copper concentration must have decreased to 40% of its initial level over this period. The group of zinc-deficient rats, which had little weight gain or copper loss, had higher tissue concentrations of the metal than did the other groups. We feel that these variations in tissue copper concentrations were more a function of body size of the animals than of zinc status per se. A copper concentration of 5 ppm is recommended for laboratory rat diets (12). In summary, rats fed diets containing 14 or 57 ppm Zn gained weight at identical rates, but the group fed at the higher zinc level accumulated twice as much zinc over the 8 weeks of balance trials. This suggests to us that homeostatically controlled retention of dietary zinc in the first 10 weeks of the lives of male rats may be set at a level beyond that necessary to produce maximal growth.
Acknowledgments Supported by USPHS ES 00159 and by funds supplied by Merrell Dow Pharmaceuticals, Incorporated. The authors wish to acknowledge valuable assistance in the experimental portion of the study by Charles Broge, and in the data analysis portion by James Hoadley.
References 1. E. Weigand and M. Kirchgessner, Nutr. Metabol. 20, 307 (1976). 2. E. Weigand and M. Kirchgessner, Nutr. Metabol. 20, 314 (1976). 3. E. Weigand and M. Kirchgessner, Z. Tierphysiol. Tierernaehrg. Futtermittelkde. 39, 16 (1977). 4. E. Weigand and M. Kirchgessner, Z. Tierphysiol., Tierernaehrg. Futtermittelkde. 39, 84 (1977). 5. E. Weigand and M. Kirchgessner, Z. Tierphysiol., Tierernaehrg. Funermittelkde. 39, 325 (1977). 6. E. Weigand and M. Kirchgessner, in Trace Element Metabolism in Man and Animals, vol. 3, M. Kirchgessner, ed., Arbeitskreis Tierernaehrungsforschung Weihenstephan, Freising-Weihenstephan, 1978, pp. 106-109. 7. E. Weigand and M. Kirchgessner, Biol. Trace Elem. Res. 1, 347 (1979). 8. E. Weigand and M. Kirchgessner, J. Nutr. 110, 469 (1980). 9. L. M. Klevay, H. G. Petering, and K. L. Stemmer, Environ. Sci. Technol. 5, 1196 (1971).
232
PETERING ET AL.
10. E. E. Menden, D. Brockman, H. Choudhury, and H. G. Petering, Anal. Chem. 49, 1644 (1977). 11. R. M. Forbes and M. Yohe, J. Nutr. 70, 53 (1960). 12. National Research Council, Nutrient Requirements of Laboratory Animals,No. 10, 2nd revised ed., National Academy of Sciences, Washington, DC, 1972. 13. American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies, J. Nutr. 107, 1340 (1977). 14. R. M. El-Gazzar, V. N. Finelli, J. Boiano, and H. G. Petering, Tox. Lett. 1, 227 (1978). 15. F. W. Bernhart and R. M. Tomarelli, J. Nutr. 89, 495 (1966). 16. H. G. Petering, M. A. Johnson, and K. L. Stemmer, Arch. Environ. Health 23, 93 (1971). 17. B. J. Winer, Statistical Principles in Experimental Design, 2nd ed., McGraw-Hill, New York, 1971, pp. 185-196.
Consecutive Zinc Balance Trials in Growing Rats H. G.
PETERING,*
E.
GIROUX,~
H.
CHOUDHURY,
AND E . E . M E N D E N
Institute of Environmental Health, Kettering Laboratory, University of Cincinnati Medical Center, Cincinatti, Ohio 45267 and
Merrell Research Center, Merrell Dow Pharmaceuticals, Cincinnatti, Ohio 45215 Received March 25, 1982; Accepted April 9, 1982
Abstract Rats were fed a purified egg white-based diet containing 5 ppm Cu and 2, 14, or 57 ppm Zn. Zinc and copper balances were determined for eight consecutive weekly trial periods. The zinc-deficient group almost ceased to gain weight and was in slightly negative zinc balance. Groups of rats fed 14 and 57 ppm Zn gained weight at equal rates. These groups were in strongly positive zinc balance for four weeks; thereafter, they were in slightly positive zinc balance. Over the eight week series of balances, the group fed 57 ppm Zn retained about two times as much zinc as did the group fed the diet containing 14 ppm Zn. All groups were in null or slightly negative copper balance throughout the trial. These results suggest that zinc accumulation may be homeostatically controlled to a level in excess of that needed for maximum growth. Index Entries: Zinc balance; homeostasis, of zinc; copper balance; optimal nutriture; growth rate effect, of Zn; effect of zinc on copper metabolism.
Introduction A series of reports by Weigand and Kirchgessner (1-8) has defined and analyzed factors that describe zinc utilization by the laboratory rat. Apparent retention is de9 1982 by The Hurnana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/82/6900-0221 $02.40
221
222
PETERING ET AL.
termined by analyses of total zinc in balance studies and is the difference between intake and excretion. The latter factor is composed of unabsorbed dietary zinc (fecal excretion only), inevitable metabolic losses, and excretion to maintain homeostasis (5). True absorption of zinc and the factoring of zinc excretion into its several components is assessed by radioisotopic techniques (1, 2). Balance studies typically are carried out for trial periods of 1 week, following an interval for adaptation of animals to the test diet. In the experiments reported below, we measured copper and zinc intake and excretion during eight consecutive weekly trial periods. Zinc balance varied considerably over the course of the study. One principal conclusion we reached was that rapidly growing rats may retain zinc in excess of that needed for maximum weight gain and that the accumulation of zinc slows as the rate of weight gain lessens. Thus, nutritional adequacy may be achieved at a level of dietary zinc below that which activates homeostatic mechanisms. Generalizations about zinc metabolism measured in short-term trials may be highly dependent on the age, dietary history of the test animals, and on other experimental conditions.
Methods Three purified diets that differed only in their contents of zinc acetate were prepared with the composition indicated in Table 1. They met NRC recommendations for all ingredients, except for the dietary levels of 2, 14, and 57 ppm Zn that were chosen to provide deficient, marginally sufficient, and abundant zinc nutriture, reTABLE 1 Composition of Diet" Diet component
g/kg
Egg white (spray-dried) Cornstarch Glucose Corn oil Mineral mixb Cellulose powder Oil-soluble vitamins C Water-soluble vitamins " Choline chloride
200.0 433.5 200.0 75.0 40.0 30.0 15.0 5.0 1.5
aDiet, except for vitamin and mineral additions, was prepared by Zeigler Bros., Inc., Gardners, PA. ~ Bernhart and Tomarelli (15) salt mix supplied (per kg diet): cupric sulfate(5 mg Cu), chromic acetate (2 mg Cr), ammoniummolybdate(0.5 mg Mo), sodium selenite (0.1 mg Se), and zinc acetate (2, 14, or 57 mg Zn). cPetering et al. (16).
ZINC BALANCE IN RATS
223
spectively. Sprague-Dawley male weanling rats (3 weeks old) with an initial weight range of 37-58 g were obtained from Charles River (Portgage, MI) and were housed individually in stainless steel cages under conditions designed to minimize their inadvertent exposure to metals (9). All rats were fed the diet containing 14 ppm Zn for an adaptation period of 3 weeks. At the age of 6 weeks they were allocated to three groups of 10 animals so that the mean body weight of each group was 171 g. One group was fed a diet containing 57 ppm Zn (by analysis) and one group was continued on diet containing 14 ppm Zn (by analysis). These diets were fed for 8 weeks. One group was fed a diet containing 1.1 ppm Zn for 2 weeks, then a diet containing 2.4 ppm Zn for 6 weeks; the intention had been to feed a diet of ca. 2 ppm Zn to the latter group for the entire period of 8 weeks, but metal analyses revealed an error, as indicated, in the preparation of the first batch of zinc-deficient diet. For every rat, weekly water and food intakes were recorded, as was body weight; feces were collected each day and pooled for each rat on a weekly basis. On one day in each of the 8 weeks of the experimental period, five rats from each of the three groups were fasted for 24 h, with free access to deionized water being permitted; during this time a complete collection of urine was made. Each rat was subjected every other week to this protocol. We assumed that data obtained from half of each group were representative of the whole group. This protocol was imposed in order to prevent dietary contamination of urine collections. After eight weeks of feeding the three diets, the rats were bled and subsequently killed and necropsied. Weights of liver, heart, pancreas, kidneys, and testes were recorded. Portions of these organs, plus serum, different batches of diet, and representative samples of excreta were submitted to analysis of their zinc and copper contents. Metal analysis was by atomic absorption spectroscopy (Perkin-Elmer Model 403); sample preparation and quality control for such analyses has been described (10).
Results Growth, Feed Consumption, and Feed Efficiency Growth curves for the groups of rats fed the three diets over the 3-week conditioning period and the 8 weeks of the experimental balance period are illustrated in Fig. 1. Rats fed the diet containing 14 ppm Zn maintained a constant rate of increase in body weight of 49 -+ 1 g (SEM) per week up to the end of the seventh week (fourth week of balances) when the mean weight was 367 + 9 (SEM). During the last 4 weeks of the study the weight gain was 25 -+ 4 g/week. There was no effect on weight gain for the group of rats shifted from the diet containing 14 ppm Zn to the one containing 57 ppm Zn. During the first 4 weeks on this diet these animals gained 49 -+ 3 g/week, while during the last 4 weeks they gained 22 -+ 3 g/week. Rats shifted to the Zn-deficient diet almost ceased to gain weight. During the last 4 weeks of the study this group of rats maintained a mean body weight of
224
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ET AL.
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350
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Fig. 1. Growthcurves for groups of 10 rats fed for three weeks with egg white-based diet containing 14 ppm Zn, then shifted to zinc-deficientdiet or zinc-abundantdiet, or continued on diet containing 14 ppm Zn. Vertical bars denote SEM. 197 -+ 1 g. The total mean increase of body weight of this group for the 8 weeks of the experimental period was 27 g compared to total increases of 295 and 283 g for the groups fed the 14 and the 57 ppm Zn diets, respectively. Our data for feed consumption and its efficiency were consistent with the observations of Weigand and Kirchgessner (3) that food intake of rats may be precisely defined by a function of live weight and weight gain. The groups fed diets containing 14 and 57 ppm Zn had comparable feed intakes as well as comparable body weight (BW) gains during each weekly interval. On the other hand, the feed consumption was substantially less for the group receiving the Zn-deficient diet. During the first 2 weeks the Zn-deficient group ate 39 and 41 g/100 g BW, respectively, while the other two groups consumed an average of 56 and 52 g/100 g BW for these two weekly periods. Feed consumption relative to body weight during the last 6 weeks was comparable in all groups and declined from an average of 44 g/100 g BW for the third week to an average of 35 g/100 g BW the last week. Over the first 4 weeks of the balance period, the Zn deficient group gained 10.7 g/100 g of feed consumed, while the two other groups gained more than three times this amount, namely 35 g/100 g of feed consumed. Feed efficiency values fell considerably over the last four weeks of the balance study. The Zn-deficient group had
ZINC BALANCE IN RATS
225
an efficiency value of - 1.6 g/100 g feed, while the adequate and excessive dietary zinc groups had efficiencies of 17.4 and 15.0 g/100 g of feed consumed, respectively.
Zinc and Copper lntake, Excretion, and Retention Mean values per rat per week of zinc and copper intake, excretion, and retention for each group summed over the first and second 4-week halves of the experimental period (w4-wl 1) are given in Table 2. The weekly values for intake and excretion relative to body weight for the group consuming the 14 ppm Zn diet are presented in Fig. 2 as illustrative of the time relationships in this group and in the one receiving the diet with the highest amount of zinc. In addition, the weekly retention values for each group in relation to body weight are presented in Fig. 3. The intakes of zinc and copper are obviously related to the feed consumption of each group as well as to the dietary metal content. Thus, during the 8-week balance period (w4-wl 1) the rats fed the diet containing 14 ppm of Zn ingested about 14 times the amount of Zn ingested by the rats in the Zn-deficient group. Furthermore, the group receiving 57 ppm Zn in the diet ingested 4 times the amount of the group receiving 14 ppm dietary Zn. There was, therefore, a fiftyfold range in the weekly intake of Zn among the three groups of rats. Over the 8-week experimental period copper intake of the Zn-deficient group amounted to 2.8 mg per rat, while the total intake of copper amounted to 5.7 mg per rat in the two other groups, i.e., double the amount of the Zn-deficient group. This was to be expected, since dietary levels were 5 ppm Cu in each diet, and feed consumption of the Zn-deficient group was one-half that of the two other groups. The amount of zinc or copper excreted in urine was very small in every group and never exceeded 0.2 mg Zn or 0.02 mg Cu per rat per week, even in the group receiving 57 ppm Zn in the diet. Nevertheless, urinary Zn did account for almost one-third of the total excretory value for the Zn-deficient group, which was in contrast to 5 and 1% of the total excretion values for the 14 and the 57 ppm dietary Zn groups, respectively. At each level of dietary Zn, fecal excretion of either Zn or Cu TABLE 2 Zinc and Copper Balance Group mean, a mg/rat Zn-deficient, 2 ppm Zn-adequate, 14 ppm Period
A. Zinc w4-w7 w8-wll B. Copper w4-w7 w8-wll
Zn-excessive, 57 ppm
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0.73 0.99
-0.20 -0.35
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2.97 5.71
+5.06 +2.41
32.15 32.19
20.35 30.31
+11.81 + 1.88
1.45 1.33
1.33 1.28
+0.12 +0.05
2.81 2.84
2.63 3.58
+0.18 -0.74
2.82 2.90
2.48 3.33
+ 0.34 - 0.43
aI zinc or copper intake for each 4 week period; E = the sum of weeklyfecal and urinary excretion for each 4 week period; R = I - E and is the overall retention for the sum of data from each of 4 weeks. =
226
PETERING ET AL.
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Fig. 2. Weekly consumption of dietary zinc (filled circles), fecal zinc excretion (open circles), and urinary zinc excretion (filled squares), for 10 rats fed a diet containing 14 ppm Zn. Group mean values are expressed per kg body weight; over 8 weeks the animals grew from a mean weight of 171 to 466 g. Vertical bars denote SEM. was the major route of excretion of these metals. There was no consistent pattern of variation with time for the fecal Zn excretion of the rats in the Zn-deficient group, but there was a progressive increase in fecal zinc in the two other groups. The same pattern was found for copper fecal excretion, namely, no consistent time variation for the Zn-deficient dietary group, but a consistent increase in weekly fecal copper values for the other two groups. The group of rats receiving the Zn-deficient diet was consistently in slightly negative Zn balance, which was somewhat greater in the second half of the balance period than in the first. Rats fed 14 ppm dietary zinc retained 63% of the zinc they ingested in the first 4 weeks of trial periods and 30% of ingested zinc in the last 4 weeks. Rats fed 57 ppm dietary zinc retained 39 and 6% of ingested zinc in the same two intervals, respectively. The 57 ppm Zn group retained more than twice as much zinc during the first half of the balance trials as did the group consuming the 14 ppm Zn diet. The 57 ppm Zn group retained slightly less than twice as much Zn as the 14 ppm Zn group for the entire 8-week period. The highest weekly retention in proportion to BW for both the 14 and 57 ppm Zn groups occurred in the first week. Thereafter, the retention values declined until the fifth week of balances, after which relatively stable positive values were recorded. It was at this point, namely, week 8, that these two groups changed their growth patterns. Over the 8-week balance period the group fed the diet with the highest content of zinc retained 21% of its intake, while the group fed the 14 ppm Zn diet retained 47% of its
ZINC BALANCE IN RATS
227
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Fig. 3. Weekly zinc balances for groups of 10 rats fed egg white-based diets containing zinc concentrations characterized as deficient, adequate, or abundant. Zinc retention is expressed per kg body weight; the zinc-deficient group increased from a mean body weight of 171 to 197 g over the 8-week period, whereas the two other groups grew to about 460 g. intake. Thus, these two groups, which did not differ substantially in either growth rate or feed consumption, did differ markedly in their retention of Zn. Copper retention in the group consuming the Zn-deficient diet varied weekly between slightly negative and slightly positive values, but the mean absolute retention values per rat in both halves of the experiment were slightly positive. In the cases of the two other groups, the copper excreted over the 8 week period was 115% and 102% of that consumed for the 14 and 57 ppm Zn groups, respectively. In both groups, retention was slightly positive in the first half and slightly negative in the second half of the experiment.
Tissue Zinc and Copper Concentrations Tissue metal data presented in Table 3 were obtained at the end of the experiment. The concentrations of zinc in kidney, pancreas, serum, and testes of the group receiving the Zn-deficient diet were statistically lower than those of the two other groups. There was a statistically significant difference between the kidney concentrations of zinc in the two groups receiving the higher dietary levels of Zn. A dose-
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response relationship appeared to hold between Zn intake and concentration in kidney, liver, and pancreas. The only effect that varying dietary zinc had on tissue copper concentrations was evident in testes, where it was found that the copper concentration of the testes of the group receiving 2 ppm dietary Zn was much higher than the values for the two other groups. The specific weights of the organs examined, i.e., the weight of the organ relative to body weight, were quite similar, as might be expected.
Discussion This experiment presents a study of the effects of feeding diets varying twentyfivefold in Zn concentration on zinc and copper balance in growing rats over a longer period of time than has usually been studied. These balances were observed during a transition from a younger physiologic state to a more mature one, as is shown by the changes that occurred in growth rates and feed intakes of the rats after the tenth week of life. Because the trials were conducted over an 8-week period, we had no reliable and useful method for continuous evaluation of true absorption and intestinal excretion of either zinc or copper. Our data, therefore, relate to balance patterns under our experimental conditions where fecal values include unabsorbed dietary zinc and copper, together with intestinally excreted zinc and copper. If weight gain is used as a criterion of nutritional adequacy, our test diets containing 14 and 57 ppm Zn would be judged equivalent. This conclusion is in line with results of Forbes and Yohe (11), who showed that 5-week weight gain of young rats is maximized when casein- or egg white-based diets contain 12 ppm Zn or greater amounts. Furthermore, the National Research Council has established (12) that a purified casein-based diet containing 12 ppm Zn meets known nutritional requirements of the rat. We labeled as abundant zinc the diet containing 57 ppm Zn. This was simply a relative designation as this level of zinc is about twice the 30 ppm level recommended for purified diets by the American Institute of Nutrition (13), but as the data show, it was four times that required under our conditions for optimal growth. An important concept in the model of homeostatic regulation of zinc metabolism is that the organism does not retain metal in excess of an optimum level that contributes to body maintenance and productive function at the time of its deposition (8). Thus, our observation that rats fed the diet containing 57 ppm Zn accumulated two times as much zinc over the 8 weeks of balance trials as did rats fed the diet containing 14 ppm Zn, implies that the latter diet was not optimal for all needs. Weigand and Kirchgessner (6), using a casein-based diet fed to weanling rats, showed in 6 day balance trials that apparent retention of zinc reached a plateau when dietary zinc concentration was 39 ppm or greater; at 10.6 ppm, apparent retention of zinc was less than half the plateau level. Our results are consistent with theirs on this point. However, Weigand and Kirchgessner found that the group fed a diet containing 10.6 ppm Zn had a mean growth rate 77% of that of the group fed at the higher level (8). The maximum weight gain of their rats was 6.2 g/day,
230
P E T E R I N G ET A L .
slightly less than the gain of 7 g/day we observed in the groups of rats fed diets containing 14 and 57 ppm Zn. The rats achieving homeostasis in their trials were in strongly positive zinc balance (5). Elsewhere (7), these authors calculated that rats of 200 g body weight would display an irreducible fecal elimination of endogenous zinc of 11-13 ~g/day. During the last 4 weeks of our study, the group of zincdeficient rats exhibited a mean negative balance of 12.5 p~g Zn/day. In principle, tissue metal concentrations indicate homeostatic balance as well, and might be employed to complement retention data. The inadequacy of the zincdeficient diet may be illustrated on this basis, since tissue zinc concentrations in rats fed this diet were significantly below those recorded in rats fed the diet containing 57 ppm Zn. The extra zinc retained by rats fed the zinc-abundant diet, compared to those fed 14 ppm dietary zinc, may be calculated to have raised wholebody zinc concentration by 6 mg/kg; and although tissue zinc concentrations that we determined were almost uniformly lower in the group fed at the lower level of dietary zinc, analytical imprecision limited us in assigning statistical significance to the differences. Weigand and Kirchgessner (4) also reported that deposition of zinc per gram of live weight gain is reflected better in whole body zinc than in zinc concentrations in liver, kidney, pancreas, and small intestine. A large amount of body zinc is found in bones, as indicated by the report of E1-Gazzar et al. (14). An inference that can be drawn from these observations is that a diet adequate for growth may not be an optimal one, if optimal is defined by activation of homeostatic regulatory mechanisms. By this definition, maximum growth rate may be insufficient as an indicator of optimal nutriture. It is unclear what functions beyond maximum growth require the increment in zinc that is the difference between adequate and optimal levels. Whatever these functions may be, they appear to be transitory: halfway through the 8 weeks of balance trials, i.e., at 10 weeks of age, the group of rats fed at the 57 ppm level of dietary zinc went into almost null balance with respect to this metal. Interestingly, the group of rats fed the 14 ppm Zn diet containing an adequate amount of zinc for growth went into a similar balance with respect to zinc at this point. Prior to this time, the latter group accumulated only half the zinc accumulated by the former group. One interpretation of this observation is that retention is homeostatically regulated to provide an excess of zinc over that necessary for all immediate metabolic requirements. This possibility is considered in the model of Weigand and Kirchgessner, but is hypothesized to occur only in special situations such as during pregnancy or disturbed metal utilization (5). In their model there is no inconsistency between adequate and optimal nutriture: dietary intake that just permits homeostatic regulation to operate is considered the basal or minimal gross requirement. Further study may clarify whether superretention of zinc generally occurs. Our sequential collection of balance data illustrates that single trials may not yield enough information for understanding all of the nutritional need for Zn. In particular, we have the opinion that the shift from a very positive zinc balance, which appears more a function of body weight (or age) than a function of zinc accumulation, may provide a lead in furthering our understanding of zinc metabolism.
ZINC BALANCE IN RATS
231
The metabolism of zinc and copper have been linked in many ways by many investigators, so we determined copper balance during the entire experiment in all three groups of rats; despite the more than twentyfold variation in dietary Zn/Cu ratio, we did not find outstanding differences in copper metabolism. Copper balance was slightly negative in both 14 and 57 ppm Zn groups of rats. Since those groups grew from mean weights of 170 to 460 g during the trials, the whole-body copper concentration must have decreased to 40% of its initial level over this period. The group of zinc-deficient rats, which had little weight gain or copper loss, had higher tissue concentrations of the metal than did the other groups. We feel that these variations in tissue copper concentrations were more a function of body size of the animals than of zinc status per se. A copper concentration of 5 ppm is recommended for laboratory rat diets (12). In summary, rats fed diets containing 14 or 57 ppm Zn gained weight at identical rates, but the group fed at the higher zinc level accumulated twice as much zinc over the 8 weeks of balance trials. This suggests to us that homeostatically controlled retention of dietary zinc in the first 10 weeks of the lives of male rats may be set at a level beyond that necessary to produce maximal growth.
Acknowledgments Supported by USPHS ES 00159 and by funds supplied by Merrell Dow Pharmaceuticals, Incorporated. The authors wish to acknowledge valuable assistance in the experimental portion of the study by Charles Broge, and in the data analysis portion by James Hoadley.
References 1. E. Weigand and M. Kirchgessner, Nutr. Metabol. 20, 307 (1976). 2. E. Weigand and M. Kirchgessner, Nutr. Metabol. 20, 314 (1976). 3. E. Weigand and M. Kirchgessner, Z. Tierphysiol. Tierernaehrg. Futtermittelkde. 39, 16 (1977). 4. E. Weigand and M. Kirchgessner, Z. Tierphysiol., Tierernaehrg. Futtermittelkde. 39, 84 (1977). 5. E. Weigand and M. Kirchgessner, Z. Tierphysiol., Tierernaehrg. Funermittelkde. 39, 325 (1977). 6. E. Weigand and M. Kirchgessner, in Trace Element Metabolism in Man and Animals, vol. 3, M. Kirchgessner, ed., Arbeitskreis Tierernaehrungsforschung Weihenstephan, Freising-Weihenstephan, 1978, pp. 106-109. 7. E. Weigand and M. Kirchgessner, Biol. Trace Elem. Res. 1, 347 (1979). 8. E. Weigand and M. Kirchgessner, J. Nutr. 110, 469 (1980). 9. L. M. Klevay, H. G. Petering, and K. L. Stemmer, Environ. Sci. Technol. 5, 1196 (1971).
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10. E. E. Menden, D. Brockman, H. Choudhury, and H. G. Petering, Anal. Chem. 49, 1644 (1977). 11. R. M. Forbes and M. Yohe, J. Nutr. 70, 53 (1960). 12. National Research Council, Nutrient Requirements of Laboratory Animals,No. 10, 2nd revised ed., National Academy of Sciences, Washington, DC, 1972. 13. American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies, J. Nutr. 107, 1340 (1977). 14. R. M. El-Gazzar, V. N. Finelli, J. Boiano, and H. G. Petering, Tox. Lett. 1, 227 (1978). 15. F. W. Bernhart and R. M. Tomarelli, J. Nutr. 89, 495 (1966). 16. H. G. Petering, M. A. Johnson, and K. L. Stemmer, Arch. Environ. Health 23, 93 (1971). 17. B. J. Winer, Statistical Principles in Experimental Design, 2nd ed., McGraw-Hill, New York, 1971, pp. 185-196.
