9 1985 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/85/0809-0121502.60
Effect of Dietary Copper and Zinc Levels on Tissue Copper, Zinc, and Iron in Male Rats CARL L. K E E N , * N A N C Y JO GOUDEY-LEFEVRE,t
H.
REINSTEIN,
MICHAEL L E F E V R E , t , $
B O L O N N E R D A L , BARBARA O . S C H N E E M A N , AND LUCILLE S . HURLEY
Department of Nutrition, University of California, Davis, California 95616 Received March 20, 1985; Accepted April 25, 1985
ABSTRACT The interaction between dietary copper and zinc as determined by tissue concentrations of trace elements was investigated in male Sprague-Dawley rats. Animals were fed diets in a factorial design with two levels of copper (0.5, 5 p,g/g) and five levels of zinc (1, 4.5, 10, 100, 1000 p~g/g) for 42 d. In rats fed the low copper diet, as dietary zinc concentration increased, the level of copper decreased in brain, testis, spleen, heart, liver, and intestine. There was no significant effect of dietary copper on tissue zinc levels. In the zinc-deficient groups, the level of iron was higher in most tissues than in tissues from controls (5 p~g Cu, 100 p~g Zndg diet). In the copper-deficient groups, iron concentration was higher than control values only in the liver. These data show that dietary zinc affected tissue copper levels primarily w h e n dietary copper was deficient, that dietary copper had no effect on tissue zinc, and that both zinc deficiency and copper deficiency affected tissue iron levels.
"Author to whom all correspondence and reprint requests should be addressed. tPresent address: Department of Physiology, Louisiana State University Medical Center, New Orleans, Louisiana 70] 12 ,~Supported by a fellowship from the California Affiliate of the American Heart Association. Biological Trace Element Research
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Index Entries; Copper, effect on tissue Cu, Zn, and Fe on dietary; zinc, effect on tissue Cu, Zn, and Fe of dietary; trace minerals, and nutrient interactions; nutrient interactions, and trace metals; iron, effect of dietary Cu and Zn on.
INTRODUCTION A n effect of dietary zinc on tissue c o p p e r levels has b e e n r e p o r t e d by several investigators. Early studies s h o w e d that rats fed toxic levels of dietary zinc h a d lower tissue c o p p e r levels than controls (1,2). S o m e studies b u t not others, have also s h o w n that w h e n the level of dietary zinc was deficient, tissue c o p p e r concentrations t e n d e d to be h i g h e r t h a n w h e n diets were a d e q u a t e in zinc (3-6). Conversely, in s o m e studies, w h e n dietary c o p p e r w a s deficient, tissue zinc concentrations were higher t h a n w h e n c o p p e r was a d e q u a t e (6-8). Recent e x p e r i m e n t s have investigated the effect of f e e d i n g marginal levels of both dietary zinc a n d dietary c o p p e r on tissue trace e l e m e n t levels. Campbell et al. (9) r e p o r t e d that rats fed a marginal level of dietary copper with high dietary zinc h a d lower liver c o p p e r concentrations t h a n did rats fed marginal c o p p e r with a n o r m a l level of zinc. In contrast, w h e n dietary copper was sufficient, the concentration of zinc in the diet did n o t affect liver c o p p e r concentration. Recent data from several laboratories have indicated that the absolute concentration of each e l e m e n t in the diet m a y be m o r e critical t h a n the ratio b e t w e e n them. In a 4 x 4 factorial design, a s t r o n g interaction b e t w e e n dietary zinc a n d c o p p e r occurred primarily w h e n either zinc or c o p p e r was deficient in the diet; only a weak interaction was n o t e d w h e n neither e l e m e n t was deficient (10). In that s t u d y , in w h i c h the p r e g n a n t rat was u s e d as a model, the interaction m a y h a v e b e e n m a g n i f i e d by the anabolic d e m a n d s of p r e g n a n c y as indicated by a stronger interaction in fetal than in maternal tissues. Hence, the develo p i n g fetus is particularly sensitive to interactions of dietary c o p p e r a n d zinc. The p r e s e n t s t u d y was u n d e r t a k e n to d e t e r m i n e w h e t h e r a similar interaction occurs in the n o n p r e g n a n t g r o w i n g animal. Since it has been s h o w n that b o t h zinc a n d c o p p e r can affect tissue iron concentrations, we also e x a m i n e d the interactive effect of dietary copper and zinc o n tissue iron concentrations. The questions p o s e d in this s t u d y were: (1) Is the copper concentration of tissues affected by c h a n g e s in dietary zinc levels? (2) If so, is there a greater effect on tissue c o p p e r concentrations w h e n dietary c o p p e r is deficient t h a n w h e n it is sufficient? (3) Does copper deficiency significantly affect tissue zinc concentrations? (4) Are d i e t - i n d u c e d changes in the tissue concentration of one of these elements related to changes in tissue concentration of the other element?
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MATERIALS AND METHODS Male weanling Sprague-Dawley rats (120-140g) were purchased from a commercial source (Simonsen Laboratories, Gilroy, CA). All animals were individually housed in s u s p e n d e d stainless steel cages in a temperature, light, and humidity controlled room (22~ 12 h light-dark cycle, 50% humidity). The rats (n = 60) were randomly assigned to one of 10 dietary groups. The basal diet consisted of isolated spray-dried egg white, 25%; sucrose, 59.5%; corn oil, 8%; salt mix, 6%; and vitamin mix, 1.5% (including supplementary biotin). The detailed composition of the diet has been published (11). A 5 x 2 factorial design was used: dietary zinc 1, 4.5, 10, 100, or 1000 ~g/g diet and dietary copper 0.5 or 10 ~g/g diet. Control diets contained 100 ~g zinc/g and 5 ~g copper/g. The diets and distilled water were fed ad libitum. Food intake was recorded twice a week and body weight was recorded weekly. After 6 wk, the rats were anesthetized with ether, laparotomized, and the lower ribs and diaphragm were cut to allow access to the heart. Blood was collected by cardiac puncture. Liver, kidney, brain, testis, lung, thymus, spleen, heart, and gastrocnemius muscle were cleaned of adhering fatty tissue, placed in acid-washed plastic vials, and frozen for analysis. The small intestine was rinsed with physiological saline and divided into three equal sections that were placed in separate acid-washed plastic vials.and stored frozen until analysis. "Intestine I" was the segment proximal to the stomach and "Intestine III" was the distal segment. Trace elements in tissues and diets were determined by flame atomic absorption spectrophotometry (Perkin-Elmer Model 370, Norwalk, CT) by the m e t h o d of Clegg et alo (12). Recovery of trace elements by this method is 98-102%. All tissues were analyzed for zinc, copper, and iron. The plasma from these rats was analyzed to determine diet induced changes in lipoprotein metabolism; the results of the lipoprotein analysis are discussed in another paper (13). Data were analyzed by two way analysis of variance. Differences between individual treatment groups were determined by least significant diffference.
RESULTS Zinc The concentration of zinc in testis, lung, spleen, heart, kidney, liver, and intestine I, II, and III increased significantly as dietary zinc increased (Fig. 1). Dietary copper levels and the statistical zinc-copper interaction did not significantly affect tissue zinc levels. Therefore, data from the groups fed the two levels of copper were combined to evaluate the effect of dietary zinc on tissue zinc concentrations. Biological Trace Element Research
VoL 8, 1985
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The tissue that exhibited the greatest change in zinc concentration was the intestine. The groups fed 1, 4.5, 10, or 100 ~g Zn/g had lower zinc concentrations in intestine segment I than did rats given zinc at 1000 ~g/g. The concentration of zinc in intestine segment II was significantly lower in the groups fed 1 or 4.5 ~g Zn/g than in those fed 10, 100, or 1000 ~g/g. Within intestinal segment II, the groups that c o n s u m e d 1000 ~g/g had significantly higher zinc concentrations than all other groups. The groups fed I or 4.5 ~g Zn/g had significantly lower zinc concentrations in intestine s e g m e n t III than did those fed 100 or 1000 p,g/g; the rats fed 10 b~g Zn/g had significantly higher tissue concentrations than those fed 1 p,g Zn/g and lower tissue concentrations than those fed 1000 ~g Zn/g. In liver, the groups fed 1 or 4.5 ~,g Zn/g had significantly lower zinc levels than did those fed 100 or 1000 ~g Zn/g, whereas those fed 1000 bLg/g had significantly higher liver zinc concentrations than all others. Tissue Zn concentrations were higher in groups fed 10 ~g Zn/g than in those fed 1 ~g Zn/g and were lower than in groups fed 1000 ~g Zn/g. Rats fed 1000 ~g/g had significantly higher kidney zinc levels than any other group. In testis, zinc concentrations were lower in the groups fed 1 or 4.5 ~g Zn/g than in the other groups. In the spleen, zinc was lower in the I ~g/g group than in all other groups and higher in the 1000 t~g/g rats than in all other groups. The groups fed the diet containing I or 4.5 ~g/g had a significantly lower concentration of zinc in the heart than did those fed 10 or 1000 ~g Zn/g. In lung, the zinc concentration was lower in the group fed 1 or 4.5 ~g Zn/g than in those fed 10 or 100 tLg Zn/g. Zinc concent-rations of brain, thymus, and gastrocnemius muscle were not affected by diet.
Copper The concentration of copper in all tissues was significantly lower in the groups fed the copper-deficient diets than in those given the copperadequate diets (Fig. 2). In the rats fed the copper-deficient diet, tissue copper concentration of brain, testis, spleen, heart, liver, and intestine segment II became lower as dietary zinc concentrations increased. In rats fed the copper-adequate diet, this effect of dietary zinc occurred only in the brain.
Fig. 1. The effect of zinc and adequate copper (0) or deficient copper (A) in the diet on tissue zinc concentrations: (la) gastrocnemius muscle, (lb) brain, (lc) testis, (ld) lung, (le) thymus, (lf) spleen, (lg) heart, (lh) kidney, (10 liver, (lj) intestine I, (lk) intestine II, (11) intestine III. Values are means • SEM. Dietary Zn significantly affected tissue Zn levels in (lc,d) P < 0.005, (lg) P < 0.01, (lf h-l) P < 0.001. Significant differences among groups fed different levels of dietary zinc are indicated by differing capital letters. Biological Trace Element Research
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In rats fed the copper-deficient diet, copper concentrations of testis, spleen, heart, liver, and intestine s e g m e n t II were significantly lower in rats fed the high zinc diet than in those fed other concentrations of zinc. With adequate copper, k i d n e y copper was lower in rats fed 1, 4.5, or 10 ~g Zn/g than in those given 100 or 1000 ~g Zn/g. Dietary zinc concentrations did not affect k i d n e y zinc concentration w h e n the copperdeficient diet was fed.
Iron Dietary zinc concentration significantly affected iron concentrations of all tissues except gastrocnemius muscle and intestine segment I (Fig. 3). In contrast, dietary copper concentration affected only liver iron. In all tissues except liver, data from the groups fed the two levels of copper were combined to evaluate the effect of dietary zinc on tissue iron concentration. In brain, the iron concentration of brain was higher in the zincdeficient group than it was in control or high zinc groups. In testis and thymus, the groups fed the zinc-deficient diets had a higher concentration of iron than did all other groups. In lung, the 4.5 and 1000 ~g Zn/g groups had lower iron concentrations than did those fed 1 or 100 ~,g/g. In spleen, the groups given 1, 4.5, or 10 ~g Zn/g had higher iron concentrations than did those fed 100 or 1000 p,g Zn/g. In heart, the group fed 1 ~,g Zn/g had higher iron concentrations than did those fed 100 ~g Zn/g. Rats fed the zinc-deficient diet had higher iron concentrations in k i d n e y than did those fed other levels of zinc. In liver, the groups fed the copper-deficient diet had significantly higher iron concentrations at 10, 100, and 1000 p,g Zn/g than the rats fed adequate copper. Rats fed zinc-deficient diets had higher liver iron concentrations than those given zinc at or above control levels. In the groups fed the control copper diet, liver iron concentration was higher w h e n zinc was 1 ~g/g than w h e n it was 10, 100, or 1000 ~g/g, and higher w h e n it was 4.5 ~g/g than w h e n it was 100 or 1000 ~g/g. Rats given 1 ~g Zn/g had higher iron concentrations in intestine segment II than those given 1000 p,g/g, and higher iron concentrations in intestine segment III than those given 4.5 ~g Zn/g.
DISCUSSION This study shows that for the male rat, dietary zinc can affect tissue copper concentration, but this antagonistic effect occurred primarily w h e n dietary copper was deficient. A n antagonistic effect of zinc on tissue copper occurred in brain tissue w h e n dietary copper was adequate. An effect of toxic levels of zinc on tissue copper concentrations has been reported w h e n control levels of dietary copper were used (1,2,14,15). Nutritionists have traditionally considered that an interaction has Biological Trace Element Research
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occurred if the tissue concentration of one n u t r i e n t changes w h e n the dietary concentration of a n o t h e r n u t r i e n t is c h a n g e d (16). This is in contrast to the statistical approach, w h e r e an interaction occurs if a c h a n g e in the dietary concentration of one e l e m e n t affects the tissue concentration of a second e l e m e n t differently at two different dietary concentrations of the second element. This p a p e r has u s e d the term interaction in the traditional nutritional view. The location of the nutritional Zn x Cu interaction is t h o u g h t to be at the level of the small intestine a n d possibly in other tissues as well. Cu § a n d Z n 2+ have the same outer electronic configuration a n d both have a coordination n u m b e r of 4. Hill a n d Matrone (16) h y p o t h e s i z e d that these elements can therefore substitute for each other in s o m e biological reactions. The cytosolic protein, metallothionein (MT), w h i c h is synthesized in response to increasing levels of intracellular zinc, binds zinc, yet the affinity of copper for the binding sites on MT is greater t h a n that of zinc (17). W h e n zinc induces MT in the small intestine, c o p p e r m a y bind to it a n d thus bring about a decrease in the absorption of copper. In s u p p o r t of the idea that the z i n c - c o p p e r interaction occurs at the level of intestinal MT, Hall et al. (7) r e p o r t e d that rats fed diets containing 3 fxg Cu/g a n d 900 txg Zn/g diet a n d given 64Cu orally had significantly higher levels of 64Cu in the MT fraction of the m u c o s a of the small intestine, a n d lower 64Cu absorption t h a n did rats fed dietary zinc at 30 ~xg/g. Hall et al. estimated that the decrease in 64Cu absorption could be acc o u n t e d for by the increase in MT b o u n d S4Cu in the small intestine. Similar results were f o u n d by Ogiso et al. (t8,19), w h o gave rats dietary zinc at 1000 ~g/g for 8 d; liver c o p p e r was lower a n d the MT fraction of the small intestine contained m o r e c o p p e r t h a n in controls. Fischer et al. (20) fed rats different levels of zinc with dietary Cu at 6 ~xg/g. Liver a n d s e r u m copper were significantly higher in g r o u p s fed 60 ~g Zn/g t h a n in those given 120 txg/g. In everted d u o d e n a l sacs from these rats, the a m o u n t of copper transferred across the mucosal cells was lower a n d the a m o u n t retained in the mucosal cell was higher in rats fed the h i g h e r concentration of zinc. Similar results were obtained with 67Cu, but the distribution of 67Cu was different (21). Most of the 67Cu in rats fed 120 ~g Z n / g was b o u n d to MT, while in rats fed 30 ~g Zn/g, m o s t of the 67Cu was in a high molecular w e i g h t fraction. Plasma and liver c o p p e r were lower in rats fed 120 Ixg Zn/g than in those fed 30 ~xg/g. These results s h o w that even if the concentration of copper in the small intestine is the same, the distribution of c o p p e r a m o n g the fractions in the small intestine m a y be different. H o w e v e r , the a m o u n t of 67Cu absorbed was the s a m e regardless of diet, w h i c h does n o t s u p p o r t the h y p o t h e s i s that d e c r e a s e d copper absorption accounts completely for the effect of dietary zinc in l o w e r i n g tissue copper levels. O n o s a k a a n d Cherian (22) p r o v i d e d information on the possibility of intermolecular interactions occurring via MT; they injected high levels of zinc into rats a n d s h o w e d a dose d e p e n d e n t increase in MT levels only in Biological Trace Element Research
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pancreas, liver, small intestine, and kidney. Our results are in a g r e e m e n t with theirs, since there was a m u c h greater increase in tissue zinc levels in small intestine, liver, and kidney than in the other tissues m e a s u ~ d . In our experiment, the small intestine had the highest concentration of zinc, whereas in Onosaka and Cherian's work, pancreas and liver were highest in zinc. This difference between the results may be because oral zinc w o u l d reach the small intestine first, whereas in their experiment, injected zinc was given and w o u l d reach the liver and pancreas before the small intestine. In both studies the increase in kidney zinc was the lowest of the three tissues. We also found that deficient and marginal levels of zinc affected the concentration of zinc in testis, lung, spleen, and heart. The concentrations of zinc in liver, intestine, and kidney were higher than in the other tissues, which changed relatively little as dietary zinc concentration increased. This w o u l d suggest that mechanisms of absorption, turnover rates, V~ax and/or binding ligands may be different in the two groups of tissues. The decrease in tissue copper concentration in the copper-deficient groups as dietary levels of zinc increased did not occur solely in those tissues most responsive to increasing dietary zinc levels; thus the decrease appears not to be a specific tissue Cu x Zn interaction. The mechanisms that make copper concentrations in certain tissues especially sensitive to dietary zinc concentration are not known. Kidney copper was highest in the groups fed adequate copper levels and control or high levels of dietary zinc. W h e n dietary copper was deficient, copper levels in the kidney were low regardless of k i d n e y zinc concentration. The same effect was seen during pregnancy (10). Thus, kidney binds copper w h e n dietary zinc and copper are sufficient. Similarly, Bremner et al. (5) s h o w e d that there was more MT in the k i d n e y in zinc-sufficient than in zinc-deficient rats~ Low tissue copper levels can be functionally significant as demonstrated by the recent work of L'Abbe and Fisher (23), who found that low copper concentrations in liver and heart resulting from high dietary zinc were accompanied by low activity of cytochrome C oxidase and Cu,Zn-superoxide dismutase. Tissue iron was significantly affected by zinc in most of the tissues examined, whereas dietary copper affected tissue iron concentrations only in liver, suggesting that the mechanisms by which copper and zinc affect tissue iron concentration are different. Because tissue zinc concentrations did not differ in some of the tissues in which iron was affected, the m e c h a n i s m by which zinc affects iron may be via intestinal absorption or subsequent transport. There is evidence that zinc a n d iron can compete for absorption in the gut (24,25). Also there may be increased binding of iron to ferritin in the zinc-deficient animal, since zinc m a y reduce the binding of iron to ferritin (26). Copper, on the other hand, seems to act mainly by facilitating the removal of iron from the liver. Alt h o u g h it has been suggested that this is caused primarily by a reduction Biological Trace Element Research
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in the plasma c o p p e r e n z y m e ferroxidase I (27), several other factors m a y be involved (28). Regardless of the m e c h a n i s m s by w h i c h deficiencies of zinc a n d c o p p e r affect iron, it is e v i d e n t that iron metabolism s h o u l d be c o n s i d e r e d in studies evaluating the interaction of dietary zinc a n d copper. This is especially i m p o r t a n t in studies of pathological effects, since high tissue iron m a y result in oxidative stress (29,30).
ACKNOWLEDGMENTS This w o r k was s u p p o r t e d in part by National Institutes of H e a l t h research grant HD-01743 f r o m the National Institute of Child H e a l t h a n d Human Development.
REFERENCES 1. S. E. Smith and E. J. Larson, J. Biol. Chem. 163, 29 (1946). 2. D. R. Grant-Frost and E. J. Underwood, Aust. J. Exp. Biol. Med. Sci. 36, 339 (1958). 3. L. Murthy, L. M. Klevay, and H. G. Petering, J. Nutr. 104, t458 (1974). 4. J. G. Reinhold, G. A. Kfoury, and To A. Thomas, J. Nutr. 92, 173 (1967). 5. [. Bremner and N. T. Davies, J. Nutr. 36, 101 (1976). 6. B. L. O'Dell, P. G. Reeves, and R. E. Morgan, in Trace Substances in Environmental Health-X, D. D. Hemphill, ed~ Univ. of Missouri, Columbia, MO, 1976, pp. 411-421. 7. A. C. Hall, B. W. Young, and I. Bremner, J. Inorg~ Biochem. 11, 57 (1979). 8. F. J. Schwarz and M. Kirchgessner, Zbl. Vet. Med. A26, 493 (1979). 9. J. K. Campbell and C. F. Mills, Proc. Nutr. Soc. 33, 15A (1974). 10. N. H. Reinstein, B. Lonnerdal, C. L. Keen, and L. S. Hurley, ]. Nutr. 114, 1266 (1984). 11. D. G. Masters, C. L. Keen, B. Lonnerdal, and L. S. Hurley, J. Nutr. 113, 905 (1983). 12. M. S. Clegg, C. L. Keen, B. Lonnerdal, and L. S. Hurley, Biol. Trace Element Res. 3, 107 (1981). 13. M. Lefevre, C. L. Keen, B. Lonnerdal, L. S. Hurley, and B. O. Schneeman, J. Nutr. 115, 359 (1985). 14. D. H. Cox and D. L. Harris, J. Nutr. 70, 514 (1960). 15. D. H. Cox, S. A. Schlicker, and R. C. Chu, J. Nutr. 98, 459 (1969). 16. C. H. Hill and G. Matrone, Fed. Proc. 29, 1481 (1970). 17. I. Bremner, Wld. Rev. Nutr. Diet. 32, 165 (1978). 18. I. Ogiso, K. Moriyama, S. Sasaki, Y. Ishirmura, and A. Miwato, Chem. Pharm. Bull. 22, 55 (1974). 19. T. Ogiso, N. Ogawa, and T. Muira, Chem. Pharm. Bull. 27, 515 (1979). 20. P. W. F. Fischer, A. Giroux, and M. R. L'Abbe, J. Clin. Nutr. 34, 1670 (1981). 21. P. W. F. Fischer, A. Giroux, and M. R. L'Abbe, ]. Nutr. 113, 462 (1983). 22. S. Onosaka and M. G. Cherian, Toxicol. 23, 11 (1982). 23. M. R. L'Abbe and P. W. F. Fischer, ]. Nutr. 114, 813 (1984). 24. C. T. Settlemire and G. Matrone, ]. Nutr. 92, 153 (1967). 25. W. W. Solomons and R. A. Jacob, Am. J. Clin. Nutr. 34, 475 (1981). 26. A. Treffey and P. M. Harrison, The Biochemistry and Physiology of Iron, P. Biological TraceElementResearch
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27. 28. 29. 30.
Saltman and J. Hegenauer, eds., Elsevier Biomedical, New York, 1982, pp. 459-461. S. Osaki, D. A. Johnson, and E. Frieden, J. Biol. Chem. 241, 2746 (1966). N. L. Cohen, C. L. Keen, B. Lonnerdal, and L. S. Hurley, BBRC 113, 127 (1983). E. D. Wills, Biochem. Pharmacol. 21, 239 (1972). A. Valenzuela, V. Fernandez, and L. Ao Videla, Toxicol. Appl. Pharmacol. 70, 87 (1983).
Biological Trace Element Research
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Effect of Dietary Copper and Zinc Levels on Tissue Copper, Zinc, and Iron in Male Rats CARL L. K E E N , * N A N C Y JO GOUDEY-LEFEVRE,t
H.
REINSTEIN,
MICHAEL L E F E V R E , t , $
B O L O N N E R D A L , BARBARA O . S C H N E E M A N , AND LUCILLE S . HURLEY
Department of Nutrition, University of California, Davis, California 95616 Received March 20, 1985; Accepted April 25, 1985
ABSTRACT The interaction between dietary copper and zinc as determined by tissue concentrations of trace elements was investigated in male Sprague-Dawley rats. Animals were fed diets in a factorial design with two levels of copper (0.5, 5 p,g/g) and five levels of zinc (1, 4.5, 10, 100, 1000 p~g/g) for 42 d. In rats fed the low copper diet, as dietary zinc concentration increased, the level of copper decreased in brain, testis, spleen, heart, liver, and intestine. There was no significant effect of dietary copper on tissue zinc levels. In the zinc-deficient groups, the level of iron was higher in most tissues than in tissues from controls (5 p~g Cu, 100 p~g Zndg diet). In the copper-deficient groups, iron concentration was higher than control values only in the liver. These data show that dietary zinc affected tissue copper levels primarily w h e n dietary copper was deficient, that dietary copper had no effect on tissue zinc, and that both zinc deficiency and copper deficiency affected tissue iron levels.
"Author to whom all correspondence and reprint requests should be addressed. tPresent address: Department of Physiology, Louisiana State University Medical Center, New Orleans, Louisiana 70] 12 ,~Supported by a fellowship from the California Affiliate of the American Heart Association. Biological Trace Element Research
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Index Entries; Copper, effect on tissue Cu, Zn, and Fe on dietary; zinc, effect on tissue Cu, Zn, and Fe of dietary; trace minerals, and nutrient interactions; nutrient interactions, and trace metals; iron, effect of dietary Cu and Zn on.
INTRODUCTION A n effect of dietary zinc on tissue c o p p e r levels has b e e n r e p o r t e d by several investigators. Early studies s h o w e d that rats fed toxic levels of dietary zinc h a d lower tissue c o p p e r levels than controls (1,2). S o m e studies b u t not others, have also s h o w n that w h e n the level of dietary zinc was deficient, tissue c o p p e r concentrations t e n d e d to be h i g h e r t h a n w h e n diets were a d e q u a t e in zinc (3-6). Conversely, in s o m e studies, w h e n dietary c o p p e r w a s deficient, tissue zinc concentrations were higher t h a n w h e n c o p p e r was a d e q u a t e (6-8). Recent e x p e r i m e n t s have investigated the effect of f e e d i n g marginal levels of both dietary zinc a n d dietary c o p p e r on tissue trace e l e m e n t levels. Campbell et al. (9) r e p o r t e d that rats fed a marginal level of dietary copper with high dietary zinc h a d lower liver c o p p e r concentrations t h a n did rats fed marginal c o p p e r with a n o r m a l level of zinc. In contrast, w h e n dietary copper was sufficient, the concentration of zinc in the diet did n o t affect liver c o p p e r concentration. Recent data from several laboratories have indicated that the absolute concentration of each e l e m e n t in the diet m a y be m o r e critical t h a n the ratio b e t w e e n them. In a 4 x 4 factorial design, a s t r o n g interaction b e t w e e n dietary zinc a n d c o p p e r occurred primarily w h e n either zinc or c o p p e r was deficient in the diet; only a weak interaction was n o t e d w h e n neither e l e m e n t was deficient (10). In that s t u d y , in w h i c h the p r e g n a n t rat was u s e d as a model, the interaction m a y h a v e b e e n m a g n i f i e d by the anabolic d e m a n d s of p r e g n a n c y as indicated by a stronger interaction in fetal than in maternal tissues. Hence, the develo p i n g fetus is particularly sensitive to interactions of dietary c o p p e r a n d zinc. The p r e s e n t s t u d y was u n d e r t a k e n to d e t e r m i n e w h e t h e r a similar interaction occurs in the n o n p r e g n a n t g r o w i n g animal. Since it has been s h o w n that b o t h zinc a n d c o p p e r can affect tissue iron concentrations, we also e x a m i n e d the interactive effect of dietary copper and zinc o n tissue iron concentrations. The questions p o s e d in this s t u d y were: (1) Is the copper concentration of tissues affected by c h a n g e s in dietary zinc levels? (2) If so, is there a greater effect on tissue c o p p e r concentrations w h e n dietary c o p p e r is deficient t h a n w h e n it is sufficient? (3) Does copper deficiency significantly affect tissue zinc concentrations? (4) Are d i e t - i n d u c e d changes in the tissue concentration of one of these elements related to changes in tissue concentration of the other element?
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MATERIALS AND METHODS Male weanling Sprague-Dawley rats (120-140g) were purchased from a commercial source (Simonsen Laboratories, Gilroy, CA). All animals were individually housed in s u s p e n d e d stainless steel cages in a temperature, light, and humidity controlled room (22~ 12 h light-dark cycle, 50% humidity). The rats (n = 60) were randomly assigned to one of 10 dietary groups. The basal diet consisted of isolated spray-dried egg white, 25%; sucrose, 59.5%; corn oil, 8%; salt mix, 6%; and vitamin mix, 1.5% (including supplementary biotin). The detailed composition of the diet has been published (11). A 5 x 2 factorial design was used: dietary zinc 1, 4.5, 10, 100, or 1000 ~g/g diet and dietary copper 0.5 or 10 ~g/g diet. Control diets contained 100 ~g zinc/g and 5 ~g copper/g. The diets and distilled water were fed ad libitum. Food intake was recorded twice a week and body weight was recorded weekly. After 6 wk, the rats were anesthetized with ether, laparotomized, and the lower ribs and diaphragm were cut to allow access to the heart. Blood was collected by cardiac puncture. Liver, kidney, brain, testis, lung, thymus, spleen, heart, and gastrocnemius muscle were cleaned of adhering fatty tissue, placed in acid-washed plastic vials, and frozen for analysis. The small intestine was rinsed with physiological saline and divided into three equal sections that were placed in separate acid-washed plastic vials.and stored frozen until analysis. "Intestine I" was the segment proximal to the stomach and "Intestine III" was the distal segment. Trace elements in tissues and diets were determined by flame atomic absorption spectrophotometry (Perkin-Elmer Model 370, Norwalk, CT) by the m e t h o d of Clegg et alo (12). Recovery of trace elements by this method is 98-102%. All tissues were analyzed for zinc, copper, and iron. The plasma from these rats was analyzed to determine diet induced changes in lipoprotein metabolism; the results of the lipoprotein analysis are discussed in another paper (13). Data were analyzed by two way analysis of variance. Differences between individual treatment groups were determined by least significant diffference.
RESULTS Zinc The concentration of zinc in testis, lung, spleen, heart, kidney, liver, and intestine I, II, and III increased significantly as dietary zinc increased (Fig. 1). Dietary copper levels and the statistical zinc-copper interaction did not significantly affect tissue zinc levels. Therefore, data from the groups fed the two levels of copper were combined to evaluate the effect of dietary zinc on tissue zinc concentrations. Biological Trace Element Research
VoL 8, 1985
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The tissue that exhibited the greatest change in zinc concentration was the intestine. The groups fed 1, 4.5, 10, or 100 ~g Zn/g had lower zinc concentrations in intestine segment I than did rats given zinc at 1000 ~g/g. The concentration of zinc in intestine segment II was significantly lower in the groups fed 1 or 4.5 ~g Zn/g than in those fed 10, 100, or 1000 ~g/g. Within intestinal segment II, the groups that c o n s u m e d 1000 ~g/g had significantly higher zinc concentrations than all other groups. The groups fed I or 4.5 ~g Zn/g had significantly lower zinc concentrations in intestine s e g m e n t III than did those fed 100 or 1000 p,g/g; the rats fed 10 b~g Zn/g had significantly higher tissue concentrations than those fed 1 p,g Zn/g and lower tissue concentrations than those fed 1000 ~g Zn/g. In liver, the groups fed 1 or 4.5 ~,g Zn/g had significantly lower zinc levels than did those fed 100 or 1000 ~g Zn/g, whereas those fed 1000 bLg/g had significantly higher liver zinc concentrations than all others. Tissue Zn concentrations were higher in groups fed 10 ~g Zn/g than in those fed 1 ~g Zn/g and were lower than in groups fed 1000 ~g Zn/g. Rats fed 1000 ~g/g had significantly higher kidney zinc levels than any other group. In testis, zinc concentrations were lower in the groups fed 1 or 4.5 ~g Zn/g than in the other groups. In the spleen, zinc was lower in the I ~g/g group than in all other groups and higher in the 1000 t~g/g rats than in all other groups. The groups fed the diet containing I or 4.5 ~g/g had a significantly lower concentration of zinc in the heart than did those fed 10 or 1000 ~g Zn/g. In lung, the zinc concentration was lower in the group fed 1 or 4.5 ~g Zn/g than in those fed 10 or 100 tLg Zn/g. Zinc concent-rations of brain, thymus, and gastrocnemius muscle were not affected by diet.
Copper The concentration of copper in all tissues was significantly lower in the groups fed the copper-deficient diets than in those given the copperadequate diets (Fig. 2). In the rats fed the copper-deficient diet, tissue copper concentration of brain, testis, spleen, heart, liver, and intestine segment II became lower as dietary zinc concentrations increased. In rats fed the copper-adequate diet, this effect of dietary zinc occurred only in the brain.
Fig. 1. The effect of zinc and adequate copper (0) or deficient copper (A) in the diet on tissue zinc concentrations: (la) gastrocnemius muscle, (lb) brain, (lc) testis, (ld) lung, (le) thymus, (lf) spleen, (lg) heart, (lh) kidney, (10 liver, (lj) intestine I, (lk) intestine II, (11) intestine III. Values are means • SEM. Dietary Zn significantly affected tissue Zn levels in (lc,d) P < 0.005, (lg) P < 0.01, (lf h-l) P < 0.001. Significant differences among groups fed different levels of dietary zinc are indicated by differing capital letters. Biological Trace Element Research
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In rats fed the copper-deficient diet, copper concentrations of testis, spleen, heart, liver, and intestine s e g m e n t II were significantly lower in rats fed the high zinc diet than in those fed other concentrations of zinc. With adequate copper, k i d n e y copper was lower in rats fed 1, 4.5, or 10 ~g Zn/g than in those given 100 or 1000 ~g Zn/g. Dietary zinc concentrations did not affect k i d n e y zinc concentration w h e n the copperdeficient diet was fed.
Iron Dietary zinc concentration significantly affected iron concentrations of all tissues except gastrocnemius muscle and intestine segment I (Fig. 3). In contrast, dietary copper concentration affected only liver iron. In all tissues except liver, data from the groups fed the two levels of copper were combined to evaluate the effect of dietary zinc on tissue iron concentration. In brain, the iron concentration of brain was higher in the zincdeficient group than it was in control or high zinc groups. In testis and thymus, the groups fed the zinc-deficient diets had a higher concentration of iron than did all other groups. In lung, the 4.5 and 1000 ~g Zn/g groups had lower iron concentrations than did those fed 1 or 100 ~,g/g. In spleen, the groups given 1, 4.5, or 10 ~g Zn/g had higher iron concentrations than did those fed 100 or 1000 p,g Zn/g. In heart, the group fed 1 ~,g Zn/g had higher iron concentrations than did those fed 100 ~g Zn/g. Rats fed the zinc-deficient diet had higher iron concentrations in k i d n e y than did those fed other levels of zinc. In liver, the groups fed the copper-deficient diet had significantly higher iron concentrations at 10, 100, and 1000 p,g Zn/g than the rats fed adequate copper. Rats fed zinc-deficient diets had higher liver iron concentrations than those given zinc at or above control levels. In the groups fed the control copper diet, liver iron concentration was higher w h e n zinc was 1 ~g/g than w h e n it was 10, 100, or 1000 ~g/g, and higher w h e n it was 4.5 ~g/g than w h e n it was 100 or 1000 ~g/g. Rats given 1 ~g Zn/g had higher iron concentrations in intestine segment II than those given 1000 p,g/g, and higher iron concentrations in intestine segment III than those given 4.5 ~g Zn/g.
DISCUSSION This study shows that for the male rat, dietary zinc can affect tissue copper concentration, but this antagonistic effect occurred primarily w h e n dietary copper was deficient. A n antagonistic effect of zinc on tissue copper occurred in brain tissue w h e n dietary copper was adequate. An effect of toxic levels of zinc on tissue copper concentrations has been reported w h e n control levels of dietary copper were used (1,2,14,15). Nutritionists have traditionally considered that an interaction has Biological Trace Element Research
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occurred if the tissue concentration of one n u t r i e n t changes w h e n the dietary concentration of a n o t h e r n u t r i e n t is c h a n g e d (16). This is in contrast to the statistical approach, w h e r e an interaction occurs if a c h a n g e in the dietary concentration of one e l e m e n t affects the tissue concentration of a second e l e m e n t differently at two different dietary concentrations of the second element. This p a p e r has u s e d the term interaction in the traditional nutritional view. The location of the nutritional Zn x Cu interaction is t h o u g h t to be at the level of the small intestine a n d possibly in other tissues as well. Cu § a n d Z n 2+ have the same outer electronic configuration a n d both have a coordination n u m b e r of 4. Hill a n d Matrone (16) h y p o t h e s i z e d that these elements can therefore substitute for each other in s o m e biological reactions. The cytosolic protein, metallothionein (MT), w h i c h is synthesized in response to increasing levels of intracellular zinc, binds zinc, yet the affinity of copper for the binding sites on MT is greater t h a n that of zinc (17). W h e n zinc induces MT in the small intestine, c o p p e r m a y bind to it a n d thus bring about a decrease in the absorption of copper. In s u p p o r t of the idea that the z i n c - c o p p e r interaction occurs at the level of intestinal MT, Hall et al. (7) r e p o r t e d that rats fed diets containing 3 fxg Cu/g a n d 900 txg Zn/g diet a n d given 64Cu orally had significantly higher levels of 64Cu in the MT fraction of the m u c o s a of the small intestine, a n d lower 64Cu absorption t h a n did rats fed dietary zinc at 30 ~xg/g. Hall et al. estimated that the decrease in 64Cu absorption could be acc o u n t e d for by the increase in MT b o u n d S4Cu in the small intestine. Similar results were f o u n d by Ogiso et al. (t8,19), w h o gave rats dietary zinc at 1000 ~g/g for 8 d; liver c o p p e r was lower a n d the MT fraction of the small intestine contained m o r e c o p p e r t h a n in controls. Fischer et al. (20) fed rats different levels of zinc with dietary Cu at 6 ~xg/g. Liver a n d s e r u m copper were significantly higher in g r o u p s fed 60 ~g Zn/g t h a n in those given 120 txg/g. In everted d u o d e n a l sacs from these rats, the a m o u n t of copper transferred across the mucosal cells was lower a n d the a m o u n t retained in the mucosal cell was higher in rats fed the h i g h e r concentration of zinc. Similar results were obtained with 67Cu, but the distribution of 67Cu was different (21). Most of the 67Cu in rats fed 120 ~g Z n / g was b o u n d to MT, while in rats fed 30 ~g Zn/g, m o s t of the 67Cu was in a high molecular w e i g h t fraction. Plasma and liver c o p p e r were lower in rats fed 120 Ixg Zn/g than in those fed 30 ~xg/g. These results s h o w that even if the concentration of copper in the small intestine is the same, the distribution of c o p p e r a m o n g the fractions in the small intestine m a y be different. H o w e v e r , the a m o u n t of 67Cu absorbed was the s a m e regardless of diet, w h i c h does n o t s u p p o r t the h y p o t h e s i s that d e c r e a s e d copper absorption accounts completely for the effect of dietary zinc in l o w e r i n g tissue copper levels. O n o s a k a a n d Cherian (22) p r o v i d e d information on the possibility of intermolecular interactions occurring via MT; they injected high levels of zinc into rats a n d s h o w e d a dose d e p e n d e n t increase in MT levels only in Biological Trace Element Research
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pancreas, liver, small intestine, and kidney. Our results are in a g r e e m e n t with theirs, since there was a m u c h greater increase in tissue zinc levels in small intestine, liver, and kidney than in the other tissues m e a s u ~ d . In our experiment, the small intestine had the highest concentration of zinc, whereas in Onosaka and Cherian's work, pancreas and liver were highest in zinc. This difference between the results may be because oral zinc w o u l d reach the small intestine first, whereas in their experiment, injected zinc was given and w o u l d reach the liver and pancreas before the small intestine. In both studies the increase in kidney zinc was the lowest of the three tissues. We also found that deficient and marginal levels of zinc affected the concentration of zinc in testis, lung, spleen, and heart. The concentrations of zinc in liver, intestine, and kidney were higher than in the other tissues, which changed relatively little as dietary zinc concentration increased. This w o u l d suggest that mechanisms of absorption, turnover rates, V~ax and/or binding ligands may be different in the two groups of tissues. The decrease in tissue copper concentration in the copper-deficient groups as dietary levels of zinc increased did not occur solely in those tissues most responsive to increasing dietary zinc levels; thus the decrease appears not to be a specific tissue Cu x Zn interaction. The mechanisms that make copper concentrations in certain tissues especially sensitive to dietary zinc concentration are not known. Kidney copper was highest in the groups fed adequate copper levels and control or high levels of dietary zinc. W h e n dietary copper was deficient, copper levels in the kidney were low regardless of k i d n e y zinc concentration. The same effect was seen during pregnancy (10). Thus, kidney binds copper w h e n dietary zinc and copper are sufficient. Similarly, Bremner et al. (5) s h o w e d that there was more MT in the k i d n e y in zinc-sufficient than in zinc-deficient rats~ Low tissue copper levels can be functionally significant as demonstrated by the recent work of L'Abbe and Fisher (23), who found that low copper concentrations in liver and heart resulting from high dietary zinc were accompanied by low activity of cytochrome C oxidase and Cu,Zn-superoxide dismutase. Tissue iron was significantly affected by zinc in most of the tissues examined, whereas dietary copper affected tissue iron concentrations only in liver, suggesting that the mechanisms by which copper and zinc affect tissue iron concentration are different. Because tissue zinc concentrations did not differ in some of the tissues in which iron was affected, the m e c h a n i s m by which zinc affects iron may be via intestinal absorption or subsequent transport. There is evidence that zinc a n d iron can compete for absorption in the gut (24,25). Also there may be increased binding of iron to ferritin in the zinc-deficient animal, since zinc m a y reduce the binding of iron to ferritin (26). Copper, on the other hand, seems to act mainly by facilitating the removal of iron from the liver. Alt h o u g h it has been suggested that this is caused primarily by a reduction Biological Trace Element Research
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in the plasma c o p p e r e n z y m e ferroxidase I (27), several other factors m a y be involved (28). Regardless of the m e c h a n i s m s by w h i c h deficiencies of zinc a n d c o p p e r affect iron, it is e v i d e n t that iron metabolism s h o u l d be c o n s i d e r e d in studies evaluating the interaction of dietary zinc a n d copper. This is especially i m p o r t a n t in studies of pathological effects, since high tissue iron m a y result in oxidative stress (29,30).
ACKNOWLEDGMENTS This w o r k was s u p p o r t e d in part by National Institutes of H e a l t h research grant HD-01743 f r o m the National Institute of Child H e a l t h a n d Human Development.
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