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Effect of dietary zinc or copper deficiency on catalase, glutathione peroxidase and superoxide dismutase activities in rat heart
NUTRITION RESEARCH, Vol. 9, pp. 319-326, 1989 0271-5317/89 $3.00 + .00 Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.
EFFECT OF DIETARY ZINC OR COPPER DEFICIENCY ON CATALASE, GLUTATHIONE PEROXIDASE AND SUPEROXIDE DISMUTASE ACTIVITIES IN RAT HEART
William J. Bettger, Ph.D. and Tammy M. Bray*, Ph.D. Department of Nutritional Sciences, College of Biological Science, University of Guelph, Guelph, Ontario, Canada NIG 2WI
ABSTRACT
Short term, severe dietary zinc or copper deficiency has been produced in young Wistar rats. Copper deficiency results in a significantly enlarged heart per unit of body weight; zinc deficiency does not. Copper deficiency caused a significant decrease in heart zinc and copper concentrations and Cu,Zn superoxide dismutase activity. Zinc deficiency resulted in elevated heart copper concentration and catalase activity. The activities of heart Mn superoxide dismutase and glutathione peroxidase were not significantly altered by the zinc or copper deficiency. Key Words:
Zn, Cu, catalase, dismutase
glutathlone peroxidase,
superoxide
INTRODUCTION
Mammalian cells have a primary (1,2) and secondary (3) antioxidant defense system against free radicals and reactive oxygen species. The enzymes of the primary antioxidant defense system which protect against reactive oxygen species are: Cu,Zn superoxide dismutase (Cu,Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase and Se-dependent glutathione peroxidase (GSH-Px). These enzymes catalyze the destruction of 02r (Cu,Zn-SOD and Mn-SOD) and H202 (eatalase, GSH-Px) (2). Each of the enzymes requires an essential mineral element in a cofactor-like role for catalytic activity. The activities of these enzymes within mammalian cells are the result of a complex interplay of the availability of the mineral cofactor and the regulation of rates of enzyme synthesis and degradation. Tissue Cu,Zn-SOD activity has been shown to be decreased under conditions of copper deficiency which lower tissue copper levels (1,4,5). Conversely, dietary zinc deficiency does not significantly affect tissue Cu,Zn-SOD activity (1,6-8). Mn-SOD activity is decreased under conditions of dietary Mn deficiency in some, but not all, tissues that have depressed Mn concentrations (9,10) and GSH-Px activity is dependent upon tissue Se concentrations (11,12). Tissue catalase activity is not decreased by dietary iron deficiency in mammals but this is largely because tissue (non-erythrocyte) heme levels are not readily affected under these conditions (13,14). Cellular hemin concentration, at least in *Author to whom correspondence
should be addressed
319
320
W.J. BETTGERand T.M. BRAY
lower eucaryotic cells, regulates the synthesis of some catalase both the transcriptional (15) and translational (16) levels.
isoenzymes at
Recently, it has been demonstrated that, under some conditions, manipulation of the dietary intake of the mineral cofactor for an enzyme in the primary antioxidant defense system can affect the activity of other enzymes in the system. For example, there are reports that liver GSH-Px activity is decreased in Fe-deficient rats (16), liver GSH-Px (17) and catalase (i) are decreased in Cu-deficient rats and liver Cu,Zn-SOD is elevated in Mn-deficient chicks (i0). The purpose of the experiments described in this report is to measure the response of the activities of enzymes that protect against reactive oxygen species in the rat heart to dietary Zn or Cu deficiency.
METHODS AND MATERIALS Animals and Diet The feeding regimen and the diets used in this study were designed to produce severe Zn or Cu deficiency in 6 week old rats and are identical to those used in other publications (1,18,19). The semi-purified diets are based on spray-dried egg white glucose hydrate and corn oil (i). To produce dietary Zn deficiency, weanling male Wistar rats (40-45 g, approximately 3 weeks old) (Charles River, Montreal, Quebec) were fed a Zn-deficient diet (
were homogenized (5% w/v) in i00 mM mM Tris, pH 7.4 at 4~ The homogenate and the supernate assayed for GSH-Px as substrate (25,26).
Catalase activity was measured as the decomposition of hydrogen peroxide (27,28). Hearts were homogenized (10% w/v) in 0.i M potassium phosphate, pH
Zn, Cu AND ANTIOXlDANT ENZYMES
321
7.5 at 4~ The h o m o g e n a t e was c e n t r i f u g e d at 6,000 x g for 20 m i n and the supernatant treated w i t h ethanol and T r i t o n X-100 prior to analysis to give the full e x p r e s s i o n of catalase a c t i v i t y (28). In all experiments, p r o t e i n was m e a s u r e d by the m e t h o d of Lowry et al. u s i n g bovine serum a l b u m i n as a standard
(29). Statistics Data were a n a l y z e d by a general Tukey's S t u d e n t i z e d range test.
linear m o d e l l i n g
procedure
followed
by
RESULTS
Rats fed the Z n - d e f i c i e n t diet showed a severe drop in food intake w h i c h r e s u l t e d in a s i g n i f i c a n t l y lower final b o d y w e i g h t than ad l i b i t u m - f e d controls. These rats h a d s i g n i f i c a n t l y lower plasma Zn but h i g h e r plasma and heart Cu concentrations. Heart Zn c o n c e n t r a t i o n and heart w e i g h t per unit of b o d y w e i g h t were not s i g n i f i c a n t l y different than Z n - a d e q u a t e controls (Table
i). TABLE Effect
1
of d i e t a r y Zn or Cu d e f i c i e n c y on body weight, ~e~rt w e i g h t p l a s m a and heart Zn and Cu concentrations '-
Parameter
Body w e i g h t Heart w e i g h t
(g) (g/100
g bw)
and
-Zn
+ZnPF
+ZnAL
54 +_ 3 a
62 + 4 a
161 + 5 b
0.46 + 0.05 a
0.39 + 0.04 a
0.38 + 0.02 a
Plasma
Zn
(#g/ml)
0.29 + 0.02 a
1.42 + 0.02 b
1.41 + 0.02 b
Plasma
Cu (#g/ml)
1.28 + 0.03 a
1.05 + 0.02 b
1.00 + 0.01 b
Heart
Zn (#g/g dry wt.)
73.1 + 0.8 a
75.8 _+ 0.8 a
73.7 _+ 0.9 a
Heart
Cu (#g/g dry wt.)
26.6 + 1.6 a
22.5 + 1.0 h
18.0 + 0.5 c
-Cu
+CuPF
+CuAL
129 + 6 a
160 + 5 b
Body w e i g h t Heart w e i g h t
(g) (g/100
127 + 7 a g bw)
0,80 + 0.14 a
0.46 + 0.03 b
0.44 + 0.02 b
Plasma
Zn (#g/ml)
1.43 _+ 0.01 a
1.42 + 0.01 a
1.43 + 0.02 a
Plasma
Cu (#g/ml)
0,09 + 0.01 a
0.99 + 0.01 b
0.98 + 0.025
Heart
Zn (#g/g dry wt.)
68.3 + 1.01 a
74.3 + 0.60 b
73.8 + 0.665
Heart
Cu (~g/g dry wt.)
10.1 + 0.3 a
19.7 + 0.70 b
18.1 + 0.19 b
7
M e a n + SEM, N=6. V a l u e s w i t h different Tukey's test.
letter
superscripts
are s i g n i f i c a n t l y
different
by
322
W.J. BETTGERand T.M. BRAY
Rats fed the Cu-defieient diet have a s i g n i f i c a n t l y lower final body weight, p l a s m a Cu and heart Zn and Cu concentrations than Cu-adequate (adllbitum) controls. Cu d e f i c i e n c y caused a significant elevation of heart weight per unit of body weight compared to p a i r - f e d and ad libitum-fed controls, Plasma Zn was not affected by dietary Cu d e f i c i e n c y (Table i). D i e t a r y Zn d e f i c i e n c y did not significantly alter heart total SOD, Cu,ZnSOD, M n - S O D or G S H - P X activities; the elevated heart catalase a c t i v i t y found in Z n - d e f i c i e n t rats is also seen in pair-fed controls. D i e t a r y Cu deficiency did not s i g n i f i c a n t l y alter heart Mn-SOD, GSH-Px or catalase activities; however, it s i g n i f i c a n t l y lowered total SOD and Cu,Zn-SOD activity. Results are shown in Table 2.
TABLE 2 Effect
of Zn or Cu deficiency on the enzymatic defense active oxygen species in the heart*'~
Parameter #
-Zn
+ZnPF
system for
+ZnAL
SOD
(units/mg)
6.36 + 0.18 a
5.86 + 0.28 a
5.84 + 0.28 b
Cu,Zn-SOD
(units/mg)
3.06 + 0.14 a
2.87 + 0.32 a
2.49 + 0.22 a
3.30 + 0.16 a
2.98 _+ 0.14 a
3.35 + 0.32 a
459 + 24 a
366 + 14 b
488 + 32 a
16.1 + i.i a
16.3 + 0.5 a
10.7 _+ 0.55
+CuPF
+CuAL
Total
Mn-SOD
(units/mg)
GSH-Px
(nmol/min/mg)
Catalase
(#mol/min/mg)
-Cu
SOD
(units/mg)
5.21 + 0.13 a
6.57 + 0.ii b
6.55 _+ 0.18 b
Cu,Zn-SOD
(units/mg)
1.67 + 0.21 a
3.47 + 0.15 b
3.17 _+ 0.09 b
3.55 + 0.22 a
3.11 + 0.16 a
3.38 + 0.15 a
Total
Mn-SOD
(units/mg)
GSH-Px
(nmol/min/mg)
Catalase
(#mol/min/mg)
524 + 28 a
497 + 14 a
504 + 14 a
ll.1 + 0.8 a
13.1 + 0.7 a
ll.8 + 0.4 a
*Mean + SEM, N=6. ~Values w i t h different letter superscripts are s i g n i f i c a n t l y Tukey's test. #All enzyme activities are e x p r e s s e d per mg of heart p r o t e i n fraction. SOD, GSH-Px and catalase activities are m e a s u r e d from different rats.
different
by
in the assay in hearts
Zn, Cu AND ANTIOXIDANT ENZYMES
323
DISCUSSION
The factors that control tissue levels of the enzymes that protect against reactive oxygen species are not completely understood. Recently, it has been demonstrated that some pro-oxidatlve conditions can alter the activity of MnSOD (30,31), Cu,Zn-SOD (31-35), catalase (31,33-35) and GSH-Px (33,35). Both dietary Zn deficiency (18,36) and Cu deficiency (37,38) in rats have been characterized as conditions which result in increased oxidative stress and/or tissue damage; however, neither nutritional pathology produces major effects on the activities of enzymes of the heart antioxidant defense system. In severely Cu-deficient rats, which have an enlarged and pathological heart (39,40), there is a depression in Cu,Zn-SOD activity that is approximately proportional to the depression in heart Cu concentration. In spite of this, heart Mn-SOD, GSH-Px and catalase activities are unaffected. In severely Zn-deficient rats, which are characterized by a severe general malnutrition caused by a voluntary reduction in food intake, Cu,Zn-SOD, Mn-SOD and GSH-Px activities are unaffected in spite of a significant elevation of heart Cu concentration. The elevated catalase activity in the hearts of Zn-deficient rats is also present in pair-fed controls. The general unresponsiveness of the activities of the heart antioxidant enzymes to these severe nutritional stress is enigmatic. The constancy of this enzyme system may be a testimony to the importance of the system to the heart tissue. On the other hand, the inability of the heart to respond to the severe nutritional stresses with compensatory changes in the activities of individual enzymes suggests the heart may be particularly susceptible to oxidative damage under some conditions. For example, the inability of the heart to compensate for a decrease in Cu,Zn-SOD activity in dietary Cu deficiency may be a contributing factor in the heart pathology (41). The response of catalase, Cu,Zn-SOD, Mn-SOD and GSH-Px to severe dietary Zn or Cu deficiency in rat heart is different than the pattern of changes described previously for rat lung and liver under these experimental conditions (i). In the lung of Zn-deficient rats, llke in heart, there is a significant elevation in catalase activity that is apparently caused by the feed restriction associated with the deficiency; however, the elevated levels of Cu,Zn-SOD and Mn-SOD in lung are not seen in the heart. In the liver of the Zn-deficient rat, Cu,Zn-SOD, Mn-SOD and GSH-PX activities are at control levels as they are in the heart; however, liver catalase significantly declines while heart catalase significantly increases. In the lung of the Cu-deficient rat, Cu,Zn-SOD is decreased while Mn-SOD and catalase activities remain constant as in the heart but GSH-Px activity is increased in the lung but not in the heart. In the liver of the Cu-deficient rat, Cu,Zn-SOD and catalase activities decline; in the heart there is not a corresponding change in catalase activity. Tissue-specific patterns of change in nutritional stress (i), coupled with different absolute activities of these enzymes in different tissues (i), makes it particularly difficult to postulate a mechanism for the control of the components of this enzyme system in rats.
ACKNOWLEDGEMENTS
This research was supported in part by NIH grant HL33491-01. are grateful for the tecnical assistance of Ms. K. Homonko.
The authors
324
W.J. BETTGERand T.M. BRAY REFERENCES
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Hill KE, Burk RF, Lane JM. Effect of selenium depletion and repletion on plasma glutathione and glutathione-dependent enzymes in the rat. J Nutr 1987; 117:99-104.
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Cusack RP, Brown WP. Iron deficiency in rats: changes in body and organ weights, plasma proteins, hemoglobins, myoglobins and catalase. J Nutr 1965; 86:383-93.
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Richter K, Ammerer G, Hartter E, Ruis H. The effect of 6-aminolevulinate on catalase T-messenger RNA levels in 6-aminolevulinate synthase defective mutants of Saccharomyces cerevisiae. J Biol Chem 1980; 255:8019-22.
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Lee YH, Layman DK, Bell RE. Glutathione peroxidase activity in irondeficient rats. J Nutr 1981; 111:194-200.
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Jenkinson SG, Lawrence RA, Burk RF, Williams DM. Effects of copper deficiency on the activity of the selenoenzvme glutathione peroxidase and on excretion and tissue retention of 75SeO3 t2. J Nutr 1982; 112:197-204.
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Hammermueller JD, Bray TM, Bettger WJ. Effect of zinc and copper deficiency on microsomal NADPH-dependent active oxygen generation in rat lung and liver. J Nutr 1987; 117:894-901.
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Bettger WJ, Taylor CG. Effects of copper and zinc status of rats on the concentration of copper and zinc in the erythrocyte membrane. Nutr Res 1986; 6:451-7.
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Clegg MS, Keen CL, Lonnerdal B, Hurley LS. Influence of ashing techniques on the analysis of trace elements in animal tissue. Biol Trace Elem Res 1981; 3:107-15.
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Misra HP, Fridovich I. Superoxide dismutase: a photochemical augmentation assay. Arch Biochem Biophys 1977; 181:308-12.
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Prohaska JR, Gutsch DE. 1983. Development of glutathlone peroxidase activity during dietary and genetic copper deficiency. Biol Trace Elem Res 1983; 5:35-45.
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Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70:158-69.
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Oberley LW, St. Clair DK, Autor AP, Oberley TD. Increase in manganese superoxide dismutase activity in the mouse heart after X-irradiation. Arch Biochem Biophys 1987; 254:69-80.
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32.
Hass MA, Massaro D. Differences in CuZn superoxide dismutase induction in lungs of neonatal and adult rats. Am J Physiol 1987; 253:C66-70.
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Wohaieb SA, Godin DV. Starvation-related alterations in free radical tissue defense mechanisms in rats. Diabetes 1987; 36:169-73.
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Blakely SR, Slaughter L, Adkins J, Knight EV. Effects of B-carotene and retinyl palmitate on corn oil-induced superoxide dismutase and catalase in rats. J Nutr 1988; 118:152-8.
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Iritani N, Ikeda Y. Activation of catalase and other enzymes by corn oil intake. J Nutr 1982; 112:2235-9.
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Bray TM, Kubow S, Bettger WJ. Effect of dietary zinc on endogenous free radical production in rat lung microsomes. J Nutr 1986; 116:1054-60.
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Balevska PS, Russanov EM, Kassabova TA. Studies on lipid peroxidatlon rat liver by copper deficiency. Int J Biochem 1981; 12:489-93.
38.
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39.
Prohaska JR, Heller LJ. Mechanical properties of the copper-deficient heart. J Nutr 1982; 112:2142-50.
40.
Kopp SJ, Klevay 114, Feliksik JM. Physiologic and metabolic characterization of cardiomyopathy induced by chronic copper deficiency. Am J Physiol 1983; 245:H855-66.
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Accepted for publication November 8, 1988.
in
rat
EFFECT OF DIETARY ZINC OR COPPER DEFICIENCY ON CATALASE, GLUTATHIONE PEROXIDASE AND SUPEROXIDE DISMUTASE ACTIVITIES IN RAT HEART
William J. Bettger, Ph.D. and Tammy M. Bray*, Ph.D. Department of Nutritional Sciences, College of Biological Science, University of Guelph, Guelph, Ontario, Canada NIG 2WI
ABSTRACT
Short term, severe dietary zinc or copper deficiency has been produced in young Wistar rats. Copper deficiency results in a significantly enlarged heart per unit of body weight; zinc deficiency does not. Copper deficiency caused a significant decrease in heart zinc and copper concentrations and Cu,Zn superoxide dismutase activity. Zinc deficiency resulted in elevated heart copper concentration and catalase activity. The activities of heart Mn superoxide dismutase and glutathione peroxidase were not significantly altered by the zinc or copper deficiency. Key Words:
Zn, Cu, catalase, dismutase
glutathlone peroxidase,
superoxide
INTRODUCTION
Mammalian cells have a primary (1,2) and secondary (3) antioxidant defense system against free radicals and reactive oxygen species. The enzymes of the primary antioxidant defense system which protect against reactive oxygen species are: Cu,Zn superoxide dismutase (Cu,Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase and Se-dependent glutathione peroxidase (GSH-Px). These enzymes catalyze the destruction of 02r (Cu,Zn-SOD and Mn-SOD) and H202 (eatalase, GSH-Px) (2). Each of the enzymes requires an essential mineral element in a cofactor-like role for catalytic activity. The activities of these enzymes within mammalian cells are the result of a complex interplay of the availability of the mineral cofactor and the regulation of rates of enzyme synthesis and degradation. Tissue Cu,Zn-SOD activity has been shown to be decreased under conditions of copper deficiency which lower tissue copper levels (1,4,5). Conversely, dietary zinc deficiency does not significantly affect tissue Cu,Zn-SOD activity (1,6-8). Mn-SOD activity is decreased under conditions of dietary Mn deficiency in some, but not all, tissues that have depressed Mn concentrations (9,10) and GSH-Px activity is dependent upon tissue Se concentrations (11,12). Tissue catalase activity is not decreased by dietary iron deficiency in mammals but this is largely because tissue (non-erythrocyte) heme levels are not readily affected under these conditions (13,14). Cellular hemin concentration, at least in *Author to whom correspondence
should be addressed
319
320
W.J. BETTGERand T.M. BRAY
lower eucaryotic cells, regulates the synthesis of some catalase both the transcriptional (15) and translational (16) levels.
isoenzymes at
Recently, it has been demonstrated that, under some conditions, manipulation of the dietary intake of the mineral cofactor for an enzyme in the primary antioxidant defense system can affect the activity of other enzymes in the system. For example, there are reports that liver GSH-Px activity is decreased in Fe-deficient rats (16), liver GSH-Px (17) and catalase (i) are decreased in Cu-deficient rats and liver Cu,Zn-SOD is elevated in Mn-deficient chicks (i0). The purpose of the experiments described in this report is to measure the response of the activities of enzymes that protect against reactive oxygen species in the rat heart to dietary Zn or Cu deficiency.
METHODS AND MATERIALS Animals and Diet The feeding regimen and the diets used in this study were designed to produce severe Zn or Cu deficiency in 6 week old rats and are identical to those used in other publications (1,18,19). The semi-purified diets are based on spray-dried egg white glucose hydrate and corn oil (i). To produce dietary Zn deficiency, weanling male Wistar rats (40-45 g, approximately 3 weeks old) (Charles River, Montreal, Quebec) were fed a Zn-deficient diet (
were homogenized (5% w/v) in i00 mM mM Tris, pH 7.4 at 4~ The homogenate and the supernate assayed for GSH-Px as substrate (25,26).
Catalase activity was measured as the decomposition of hydrogen peroxide (27,28). Hearts were homogenized (10% w/v) in 0.i M potassium phosphate, pH
Zn, Cu AND ANTIOXlDANT ENZYMES
321
7.5 at 4~ The h o m o g e n a t e was c e n t r i f u g e d at 6,000 x g for 20 m i n and the supernatant treated w i t h ethanol and T r i t o n X-100 prior to analysis to give the full e x p r e s s i o n of catalase a c t i v i t y (28). In all experiments, p r o t e i n was m e a s u r e d by the m e t h o d of Lowry et al. u s i n g bovine serum a l b u m i n as a standard
(29). Statistics Data were a n a l y z e d by a general Tukey's S t u d e n t i z e d range test.
linear m o d e l l i n g
procedure
followed
by
RESULTS
Rats fed the Z n - d e f i c i e n t diet showed a severe drop in food intake w h i c h r e s u l t e d in a s i g n i f i c a n t l y lower final b o d y w e i g h t than ad l i b i t u m - f e d controls. These rats h a d s i g n i f i c a n t l y lower plasma Zn but h i g h e r plasma and heart Cu concentrations. Heart Zn c o n c e n t r a t i o n and heart w e i g h t per unit of b o d y w e i g h t were not s i g n i f i c a n t l y different than Z n - a d e q u a t e controls (Table
i). TABLE Effect
1
of d i e t a r y Zn or Cu d e f i c i e n c y on body weight, ~e~rt w e i g h t p l a s m a and heart Zn and Cu concentrations '-
Parameter
Body w e i g h t Heart w e i g h t
(g) (g/100
g bw)
and
-Zn
+ZnPF
+ZnAL
54 +_ 3 a
62 + 4 a
161 + 5 b
0.46 + 0.05 a
0.39 + 0.04 a
0.38 + 0.02 a
Plasma
Zn
(#g/ml)
0.29 + 0.02 a
1.42 + 0.02 b
1.41 + 0.02 b
Plasma
Cu (#g/ml)
1.28 + 0.03 a
1.05 + 0.02 b
1.00 + 0.01 b
Heart
Zn (#g/g dry wt.)
73.1 + 0.8 a
75.8 _+ 0.8 a
73.7 _+ 0.9 a
Heart
Cu (#g/g dry wt.)
26.6 + 1.6 a
22.5 + 1.0 h
18.0 + 0.5 c
-Cu
+CuPF
+CuAL
129 + 6 a
160 + 5 b
Body w e i g h t Heart w e i g h t
(g) (g/100
127 + 7 a g bw)
0,80 + 0.14 a
0.46 + 0.03 b
0.44 + 0.02 b
Plasma
Zn (#g/ml)
1.43 _+ 0.01 a
1.42 + 0.01 a
1.43 + 0.02 a
Plasma
Cu (#g/ml)
0,09 + 0.01 a
0.99 + 0.01 b
0.98 + 0.025
Heart
Zn (#g/g dry wt.)
68.3 + 1.01 a
74.3 + 0.60 b
73.8 + 0.665
Heart
Cu (~g/g dry wt.)
10.1 + 0.3 a
19.7 + 0.70 b
18.1 + 0.19 b
7
M e a n + SEM, N=6. V a l u e s w i t h different Tukey's test.
letter
superscripts
are s i g n i f i c a n t l y
different
by
322
W.J. BETTGERand T.M. BRAY
Rats fed the Cu-defieient diet have a s i g n i f i c a n t l y lower final body weight, p l a s m a Cu and heart Zn and Cu concentrations than Cu-adequate (adllbitum) controls. Cu d e f i c i e n c y caused a significant elevation of heart weight per unit of body weight compared to p a i r - f e d and ad libitum-fed controls, Plasma Zn was not affected by dietary Cu d e f i c i e n c y (Table i). D i e t a r y Zn d e f i c i e n c y did not significantly alter heart total SOD, Cu,ZnSOD, M n - S O D or G S H - P X activities; the elevated heart catalase a c t i v i t y found in Z n - d e f i c i e n t rats is also seen in pair-fed controls. D i e t a r y Cu deficiency did not s i g n i f i c a n t l y alter heart Mn-SOD, GSH-Px or catalase activities; however, it s i g n i f i c a n t l y lowered total SOD and Cu,Zn-SOD activity. Results are shown in Table 2.
TABLE 2 Effect
of Zn or Cu deficiency on the enzymatic defense active oxygen species in the heart*'~
Parameter #
-Zn
+ZnPF
system for
+ZnAL
SOD
(units/mg)
6.36 + 0.18 a
5.86 + 0.28 a
5.84 + 0.28 b
Cu,Zn-SOD
(units/mg)
3.06 + 0.14 a
2.87 + 0.32 a
2.49 + 0.22 a
3.30 + 0.16 a
2.98 _+ 0.14 a
3.35 + 0.32 a
459 + 24 a
366 + 14 b
488 + 32 a
16.1 + i.i a
16.3 + 0.5 a
10.7 _+ 0.55
+CuPF
+CuAL
Total
Mn-SOD
(units/mg)
GSH-Px
(nmol/min/mg)
Catalase
(#mol/min/mg)
-Cu
SOD
(units/mg)
5.21 + 0.13 a
6.57 + 0.ii b
6.55 _+ 0.18 b
Cu,Zn-SOD
(units/mg)
1.67 + 0.21 a
3.47 + 0.15 b
3.17 _+ 0.09 b
3.55 + 0.22 a
3.11 + 0.16 a
3.38 + 0.15 a
Total
Mn-SOD
(units/mg)
GSH-Px
(nmol/min/mg)
Catalase
(#mol/min/mg)
524 + 28 a
497 + 14 a
504 + 14 a
ll.1 + 0.8 a
13.1 + 0.7 a
ll.8 + 0.4 a
*Mean + SEM, N=6. ~Values w i t h different letter superscripts are s i g n i f i c a n t l y Tukey's test. #All enzyme activities are e x p r e s s e d per mg of heart p r o t e i n fraction. SOD, GSH-Px and catalase activities are m e a s u r e d from different rats.
different
by
in the assay in hearts
Zn, Cu AND ANTIOXIDANT ENZYMES
323
DISCUSSION
The factors that control tissue levels of the enzymes that protect against reactive oxygen species are not completely understood. Recently, it has been demonstrated that some pro-oxidatlve conditions can alter the activity of MnSOD (30,31), Cu,Zn-SOD (31-35), catalase (31,33-35) and GSH-Px (33,35). Both dietary Zn deficiency (18,36) and Cu deficiency (37,38) in rats have been characterized as conditions which result in increased oxidative stress and/or tissue damage; however, neither nutritional pathology produces major effects on the activities of enzymes of the heart antioxidant defense system. In severely Cu-deficient rats, which have an enlarged and pathological heart (39,40), there is a depression in Cu,Zn-SOD activity that is approximately proportional to the depression in heart Cu concentration. In spite of this, heart Mn-SOD, GSH-Px and catalase activities are unaffected. In severely Zn-deficient rats, which are characterized by a severe general malnutrition caused by a voluntary reduction in food intake, Cu,Zn-SOD, Mn-SOD and GSH-Px activities are unaffected in spite of a significant elevation of heart Cu concentration. The elevated catalase activity in the hearts of Zn-deficient rats is also present in pair-fed controls. The general unresponsiveness of the activities of the heart antioxidant enzymes to these severe nutritional stress is enigmatic. The constancy of this enzyme system may be a testimony to the importance of the system to the heart tissue. On the other hand, the inability of the heart to respond to the severe nutritional stresses with compensatory changes in the activities of individual enzymes suggests the heart may be particularly susceptible to oxidative damage under some conditions. For example, the inability of the heart to compensate for a decrease in Cu,Zn-SOD activity in dietary Cu deficiency may be a contributing factor in the heart pathology (41). The response of catalase, Cu,Zn-SOD, Mn-SOD and GSH-Px to severe dietary Zn or Cu deficiency in rat heart is different than the pattern of changes described previously for rat lung and liver under these experimental conditions (i). In the lung of Zn-deficient rats, llke in heart, there is a significant elevation in catalase activity that is apparently caused by the feed restriction associated with the deficiency; however, the elevated levels of Cu,Zn-SOD and Mn-SOD in lung are not seen in the heart. In the liver of the Zn-deficient rat, Cu,Zn-SOD, Mn-SOD and GSH-PX activities are at control levels as they are in the heart; however, liver catalase significantly declines while heart catalase significantly increases. In the lung of the Cu-deficient rat, Cu,Zn-SOD is decreased while Mn-SOD and catalase activities remain constant as in the heart but GSH-Px activity is increased in the lung but not in the heart. In the liver of the Cu-deficient rat, Cu,Zn-SOD and catalase activities decline; in the heart there is not a corresponding change in catalase activity. Tissue-specific patterns of change in nutritional stress (i), coupled with different absolute activities of these enzymes in different tissues (i), makes it particularly difficult to postulate a mechanism for the control of the components of this enzyme system in rats.
ACKNOWLEDGEMENTS
This research was supported in part by NIH grant HL33491-01. are grateful for the tecnical assistance of Ms. K. Homonko.
The authors
324
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Accepted for publication November 8, 1988.
in
rat