BIOLOGICAL TRACE ELEMENT RESEARCH 2, 175-191 (1980)
Investigations into Combined Dietary Deficiencies of Copper, Selenium, and Vitamin E in the Rat D. I. PAYNTER*~" "'Attwood" Veterinary Research Laboratory, Mickleham Road, Westmeadows, Victoria, Australia, 3047 AND
G. B. MARTIN Department of Animal Science and Production, University of Western Australia, Nedlands, Western Australia, 6009 Received November 6, 1979; Accepted February 23, 1980
Abstract Interactions between dietary Cu, Se, and vitamin E in ascorbate-induced hemolysis of erythrocytes obtained from rats fed diets deficient or adequate in these elements were investigated. Hemolysis was affected by all three dietary factors, through closely interrelated but distinct mechanisms. In vitamin Edeficient cells, hemolysis was increased and the amount of hemolysis was directly related to the amount of hemoglobin breakdown. Deficiency of Cu or Se decreased hemolysis, but only in vitamin E-deficient cells. Vitamin E did not affect the breakdown of hemoglobin, but Cu and Se did. Hemolysis and hemoglobin breakdown were decreased by the addition of glucose, through mechanisms independent of that involving reduced glutathione metabolism. These results suggest that vitamin E acts within erythrocyte membranes to prevent products of hemoglobin breakdown from initiating peroxidation and subsequent hemolysis. Effects of Cu and Se are linked with that of vitamin E by the involvement of glutathione peroxidase and Cu superoxide dismutase in the cytoplasmic breakdown of hemoglobin, rather than by a direct effect of ~Previous address: Department of Animal Science and Production, University of Western Australia, Nedlands, Western Australia 6009. 9 1980 The Humana Press Inc. All rights of any nature whatsoever reserved. 0163 -498,t / 80 / 06004) 175$03.40
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these enzymes on lipid peroxidation. It is concluded that the erythrocyte, because of its high heine content, probably represents a special system in terms of peroxidative pathways, and these findings may not be directly applicable to other tissues. Index Entries: Copper, interactions with Se and Vitamin E in rats; selenium, interactions with Cu and vitamin E in rats; vitamin E, interactions with Cu and Se in rats; dietary deficiencies, of Cu, Se, and vitamin E in rats; interactions of Cu, Se, and vitamin E in rats; hemolysis mechanisms, and Cu-Se-vitamin E interactions in rats.
Introduction The role of selenium (Se) in glutathione peroxidase, and its interaction with vitamin E, has been postulated to operate through the role of these elements in peroxide metabolism [Noguchi et al. (I); Hoekstra, (2)]. Although the actual in vivo mechanisms leading to peroxide formation have not been fully elucidated, it appears that superoxide (O2)or its reaction products may play a significant role in peroxide initiation [Kellogg and Fridovich (3); Tyler (4); Peterson and Aust (5)]. Hence, one of the roles postulated for the superoxide dismutase enzymes is that of the removal of O~ as a primary defense against deleterious peroxidation. Two forms of superoxide dismutase have been characterized in mammalian cells; one form contains Cu and Zu (CuSOD) [McCord and Fridovich (6)], and is predominantly cytoplasmic, while the second form contains Mn and appears at least in some species to be localized in the mitochrondria [Weisigner and Fridovich (7)], In the rat, liver and erythrocyte CuSOD activity is rapidly depleted with dietary Cu deficiency [Paynter et. al. (8]. In the liver of the rat, activity of glutathione peroxidase, which also has a predominantly cytoplasmic localization, is rapidly depleted with dietary Se deficiency [Hafeman et al. (9)]. This tissue also appears to be most susceptible to a combined Se-vitamin E deficiency in this species, and under appropriate conditions, liver necrosis is characteristic of this deficiency [Schwarz (10)]. Hence, a combined deficiency of Cu, Se, and vitamin E could render tissues such as the liver particularly susceptible to peroxide damage, through both an increased amount of peroxide formation, and a decreased rate of removal of these peroxides. Interactions between Cu and Se in which the toxicity or availability of one of these elements is reduced by increasing the dietary concentration of the other, have been reported previously [Jensen (11); Hill (12)] The mechanisms of such interactions are at a different level from those interactions that can be proposed at more physiological, and in particular,
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dietarily deficient concentrations of these elements, based on changes in apparently interacting enzyme systems. At these lower dietary concentrations, significant interactions between Cu and Se have been reported for production related parameters in sheep [Hill et al. (13); Thomson and Lawson (14)]. The possible mechanisms of these interactions have not been investigated. The aim of the experiments to be described was to determine the effects of Cu deficiency in rats deficient in Se and/or vitamin E. Emphasis was placed on the biochemical changes relating to these deficiencies, and the use of the erythrocyte as an in vitro model system, in an attempt to determine more fully the physiological significance of these dietarily-induced changes.
Materials and Methods Animals and Diets Twenty-one day old weanling male rats of the Sprague-Dawley strain were used in the experiments. These rats were derived from females fed a Sedeficient diet (as shown in Table 1, supplemented with Cu and vitamin E) from the time of mating onwards. At weaning, rats were housed individually in stainless steel and plastic cages at constant temperature (25~ and controlled lighting (lighting 0600 to 1900 hours), and feeding of the experimental diets was begun. The composition of the basal diet is shown in Table 1. Each of the three dietary variables (Cu, Se, and vitamin E) was used at two concentrations; deficient (-), and adequate (+), giving a total of eight dietary combinations. Deficient concentrations were those present in the basal diet, i.e., 0.5 mg Cu/kg and 0.025 mg Se/kg. Vitamin E content was not assayed directly, but was estimated to be less than 2 mg/kg, based on the reported vitamin E content of lard [Quaife and Harris (15)]. For the adequate diets, Cu was added (as CuSO4 9 5H20) at 10 mg Cu/kg diets, Se (as Na2SeO3) at 0.25 mg Se/kg and vitamin E (as dl-a-tocopherol acetate) at 100 mg/kg diet. Each dietary group contained 10 rats. Feed and deionized water were supplied ad libitum to all rats throughout the experiment. Feed residues were collected and weighed daily and rats were weighed weekly. Tissue Sampling Tissue samples for hemolysis and enzyme activity studies were obtained from rats that had been fed the diets for 50 days. These rats were randomly selected from those remaining in each dietary group at this time.Following light etherization, blood was obtained by cardiac puncture using heparinized syringes, and quickly chilled in ice water. Required organs were removed, washed in cold 154 mM NaC1, then placed in aluminum foil containers chilled on cracked ice.
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TABLE 1 Composition of the Basal Diet Ingredient Caseina Glucose monohydrate Salt mixb Vitamin mixc Lard Cod liver oil DL-methionine Choline chloride
g/kg diet 200 660 53.4 2.5 60 20 4 0.522
aSunnywest Diaries, Perth, Western Australia. bProvided the followingas g/kg diet: NaH2PO~.2H20, 26.0; CaCO3, 18.2; KCI,3.5; Na2CO~, 1.2;MgSO4.7H~O, 3.99; MnSO4.H20, 0.15; FeSO4'7H20, 0.249; ZnSO4.7H~O,0.132; NaF, 0.00025; (NH4)6MoTO24"4H20, 0.002; CoSO4.THzO,0.01; KI, 0.0003. CProvided the followingas mg/kg diet: thiamin-HCl, 12; niacin, 100; inositol, 100;p-aminobenzoicacid, 100; Ca pantothenate, 30; riboflavin, 15; pyridoxine-HCl, 12; folic acid, 2; menadione,1.5;biotin,0.5; cyanocobalamin, 0.03; retinyl palmitate (5 X 105 USP units/g), 6; ergocalciferol (4 X 107 USP units/g), 0.05.
Erythrocyte Hemolysis Studies The method used for the erythrocyte hemolysis study was essentially that described by Rotruck et al. (16), with some modifications. From each animal's whole blood, duplicate samples were taken each containing the equivalent of 0.30 mL packed cells (calculated from the hematocrit value for the whole blood), and added to tubes containing either 10 mL of 152 m M NaC1 + 10mM NazHPO4, pH 7.4 (PBS), or 10 mL of PBS + 11 m M glucose. After mixing and centrifugation, the supernatants were removed by aspiration, and a further 10 mL of PBS or PBS + glucose was added to the respective tubes. Ascorbate (0.1 mL of 220 m M Na ascorbate in PBS, pH 7.4) was then added and the tubes incubated at 37~ Until this incubation step, all operations were performed at 4~ For hemolysis determination, 0.5 mL samples of incubating cells were withdrawn, and added to tubes containing either 3 mL of PBS or 3 mL of water only. Following centrifugation, absorbance at the supernatant solutions was measured at 540 nm. From these absorbances, hemolysis and hemoglobin breakdown (decrease in absorbance of 540 nm, of totally hemolyzed sample, using To value as reference) were estimated. Reduced glutathione (GSH) was measured (as sulfhydryl) by the method of Beutler et al. (17) with modifications as described by Rotruck et al. (16). Samples for
COPPER-SELENIUM-VITAMIN E INTERACTIONS
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the estimation of hemolysis, hemoglobin breakdown, and GSH concentrations, were taken at 0, 1, 3, and 6 h after beginning the ascorbate incubations at 37~
Enzyme and Other Assays For measurement of glutathione peroxidase and CuSOD in heart and liver, 10% (W/V) homogenates of these tissues were prepared in 0.2% (V/V) Triton-X100, using glass-glass tissue homogenizers. For measuring activities in erythrocytes, whole blood was first centrifuged and the plasma removed. The cells were then washed three times in PBS and after the final wash, a sample of the packed cells was diluted 1 in 5 with water. Glutathione peroxidase activity was measured using the method of Paglia and Valentine (18), with modifications as described previously by Paynter (19). One unit of activity (U) is equivalent to 1 #mol of NADPH oxidized per min at 25~ CuSOD activity in chloroform-ethanol treated homogenates and lysates, was determined by the method of McCord and Fridovich (6), with minor modifications [Paynter et al. (8)]. Units of activity are as described by McCord and Fridovich (6). Vitamin E concentration in plasma was determined by the fluorimetric method of Storer (20). Plasma asparate aminotransferase activity was measured using a commercial test kit method (Boehringer Mannheim). Hemoglobin content of whole blood was determined by the cyanomethemoglobin method [Kampen and Zijlstra (21)]. Concentration of Se in diets was determined fluorimetrically [Watkinson (22)]. Cu was measured using conventional flame atomic absorption spectometry, following wet ashing of samples with nitric-perchloric acids.
Statistics Chi-square analysis was used for testing the significance of treatments on the incidence of rat deaths [Snedecor and Cochran (23)]. With all other data, differences between treatments were tested for significance using a factorial analysis [Snedecor and Cochran (23)]. Values for glutathione peroxidase activities and plasma vitamin E concentrations were logt0 transformed before analysis. Arcsine transformations were applied to the erythrocyte hemolysis data, with glucose included as an additional factor in this analysis.
Results Rats began dying after the experimental diets had been fed for 5 weeks. At this time, Cu was the only dietary factor that significantly affected bodyweight (Table 2). By 7 weeks, the increasing frequency of deaths in
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some groups, forced the termination of the experiment. All deaths were confined to the - C u diets and although more rats died when fed - C u - S e compared to - C u +Se diets (9/20 compared to 3/20) only the effects of Cu were significant. In all rats that died, post-mortem examination revealed large amounts of clotted blood in the chest cavity owing to rupture of the heart in the region of the ventricular apex. Heart ventricles were grossly enlarged with extensive fibrosis evident at the apex. In dead rats heart weights were increased up to 3-fold compared to rats of similar bodyweight fed adequate diets. In the rats that were killed at 7 weeks, only Cu significantly affected heart weights. Cu deficiency also resulted in small but significant decreases in blood hemoglobin and packed cell volumes after feeding the diets for 7 weeks. No significant effects (or interactions) of either Se or vitamin E were observed in these parameters (Table 2). In contrast, aspartate aminotransferase activities in plasma were increased in rats fed - S e and -vitamin E diets, and there were significant vitamin E X Se and Se • Cu interactions (Table 3). Activities of CuSOD and glutathione peroxidase in erythrocytes, liver, and heart tissues of rats fed the various diets for 7 weeks are shown in Table 3. In rats fed - S e diets, glutathione peroxidase activities were reduced to less than 5% that in +Se rats for all tissues assayed. Cu deficiency decreased CuSOD activity in liver, erythrocyte, and heart to 17, 25, and 43%, respectively, of control activities. Cu deficiency increased glutathione peroxidase activities in the erythrocyte and to a lesser extent in the liver; plasma vitamin E and erythrocyte GSH concentrations were both increased (Table 3; Fig. 1). In the in vitro hemolysis studies, all dietary factors significantly affected hemolysis, but the major dietary effect was associated with vitamin E. Effects of dietary Cu and Se were only observed with erythrocytes from vitamin E-deficient animals (Fig. 1A, B). Inclusion of glucose decreased the amount of hemolysis in-vitamin E erythrocytes, but did not affect the small amount of hemolysis in +vitamin E cells. Compared to the effects of vitamin E on hemolysis, the reverse was observed with dietary Se in vitamin Edeficient cells. Without glucose, -Se-vitamin E cells had less hemolysis than +Se -vitamin E cells. With added glucose this effect disappeared, and Se status of the erythrocytes did not affect hemolysis regardless of the vitamin E or Cu status of these cells. Similar effects of dietary Se on hemolysis were also observed for Cu, with cells from - C u -vitamin E rats hemolyzing less than cells from +Cu -vitamin E rats. However, in contrast to dietary Se, where effects were only observed in the absence of glucose, these effects of Cu were observed both in the presence and absence of glucose in the incubation mixture (Figs. 1A, B). Inhibition of hemolysis by glucose was in general associated with a maintenance of erythrocyte GSH concentrations (Fig. 1C, D). However, maintenance of GSH did not prevent hemolysis as evidenced by the considerable hemolysis in erythrocytes from -vitamin E +Cu rats,
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FIG. 1. Effects of dietary Cu, Se, and vitamin E on erythrocyte hemolysis and GSH concentrations in cells incubated with ascorbate, and with and without added glucose. For hemolysis (A,B), values shown are arcsin transformed means of three rats in each dietary treatment. Vertical bars represent the overall SEM at the times indicated. Significant dietary effects in the factorial analysis were as follows: At I h, E**, Se*, Cu*, glucose (G)**, ExSe**, ExCu**, ExG**. At 6 h, E**, Se*, Cu**, G**, ExSe**, ExCu**, ExG**, SexG**, ExSexG*, SexCuxG*. For reduced glutathione (GSH) concentrations (C, D), values shown are nontransformed means of three rats in each dietary treatment. Vertical bars represent the overall SEM at the time indicated. Significant dietary effects in the factorial analysis were as follows; At To, Cu**; decrease in GSH in first 3 h of incubation, Cu** (incubation without glucose). Broken lines represent +vitamin E, solid lines -vitamin E diets, e, - C u -Se; e, - C u +Se; II, + C u - S e ; . , +Cu +Se. (*, p < 0.05; **, p < 0.01) 184
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irrespective of Se and GSH status, when incubated in the presence of glucose (Fig. 1D). Without glucose in the incubation medium, there was a rapid decrease in erythrocyte GSH concentration with all cells in the first hour of incubation (Fig. 1C). These decreases continued until at least 3 h, after which GSH concentrations were generally increased. GSH increased the most in those - v i t a m i n E erythrocytes that were most intensively hemolyzed. The decreases in GSH concentration measured in the first hour of incubation without glucose were significantly greater in cells from - C u rats. These cells were also higher in GSH compared to other cells, prior to incubation (Fig. 1C, D). Neither vitamin E nor Se status of the cells significantly affected GSH concentrations, either before or in the first hour of incubation with ascorbate. As in the cells incubated with added glucose, the GSH content of cells was not related to the degree of hemolysis. Relative breakdown of hemoglobin in cells incubated without glucose for 6 h was significantly affected by dietary selenium only. In the presence of glucose, overall hemoglobin breakdown was decreased, and only dietary Cu significantly affected this breakdown. In both cases where significant dietary effects were obtained, cells from supplemented rats, i.e., +Se or +Cu, showed greater hemoglobin breakdown than cells from deficient rats (Fig. 2). Dietary vitamin E had no effect on hemoglobin breakdown. However, the amount of hemoglobin breakdown was highly correlated to the amount of hemolysis in -vitamin E erythrocytes at 6 h (Fig. 3). In these cells, a continuous correlation was apparent between hemoglobin breakdown and hemolysis. The continuous nature of this relationship was not affected by glucose status of cells or by dietary effects of Cu and Se. In +vitamin E cells, this correlation was not present, minimal hemolysis occurred in the presence of considerable hemoglobin breakdown.
Discussion In contrast to previously reported interactions between Cu and Se in the growth of sheep [Hill et al. (13); Thomson and Lawson (14)], only Cu significantly affected growth in the present experiment. At the tissue level, deficiency of Cu was associated with hypertrophy and fibrosis of the heart. The incidence of fatal heart lesions in these rats was increased 3-fold with a concurrent Se deficiency. Although hypertrophy of the heart is a consistent feature of Cu deficiency in the rat [Abraham and Evans (24)], extensive fibrosis occurring at the apex is not. Venticular fibrosis, similar to that observed in this experiment, has been described by Bennetts et al. (25) with "falling disease" in cattle, a disease responsive to Cu, but not always associated with even severe Cu deficiency [Underwood (26)]. The present results indicate that although a dominant role is played by Cu in the development of these lesions, other factors, including selenium, may also be involved.
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FIG. 3. Relationship between hemoglobin breakdown and hemolysis for erythrocytes, after 6 h incubation in the presence of ascorbate. Values shown were those obtained for individual rats, fed diets containing_+ Se and _+ Cu. Erythrocytes from +vitamin E rats, incubated with (D), and without glucose (9 Erythrocytes from -vitamin E rats, incubated with ( . ) , and without glucose (e). Liver necrosis was not observed in any of the dietary treatments even though changes in glutathione peroxidase and CuSOD activities were greatest in this tissue. This indicates that factors additional to these enzymes are involved in determining gross organ damage. These additional factors include other enzymes requiring the same dietary elements and other enzymes able to catalyze the same reactions, but not dependent on the same dietary elements for activity. An example of the former is the variable tissue effect of Cu deficiency on cytochrome oxidase activity compared with CuSOD [Paynter et al. (8)]. Examples of the latter include the manganesecontaining SOD that may compensate for changes in CuSOD activity, and the non-Se-dependent glutathione peroxidase, i.e., the glutathione transferases [Prohaska and Ganther (27)] compensating for the Sedependent enzyme. In erythrocytes, many of these complications are either minimal or absent. Thus, glutathione transferase activity is not present in these cells [ Lawrence and Burk (28)] and they do not contain mitochondrial-associated enzymes such as cytochrome oxidase. In the present experiment, several
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apparently interacting effects were observed between Cu and Se in erythrocytes. The major effect was the increases in both measured glutathione peroxidase activity and in GSH concentrations with Cu deficiency. Since the reaction rate of glutathione peroxidase with peroxide substrates is dependent on GSH concentration [Ganther et al. (29)], the potential activity of this enzyme will be significantly increased with Cu deficiency. Small increases in plasma vitamin E were also associated with Cu deficiency in the present experiment. These changes, taken together, may indicate that compensating mechanisms are operating in vivo for the decreased CuSOD activity in Cu-deficient erythrocytes. This concept was not supported by the results of the in vitro hemolysis experiment. The results of this experiment were also unexpected, based on previously postulated antioxidant functions of glutathione peroxidase and SOD. Although hemolysis increased with vitamin E deficiency, Cu and Se deficiencies both decreased hemolysis. These effects of Cu and Se were confined to vitamin E-deficient cells, in which the amount of hemolysis directly related to the amount of hemoglobin breakdown. Hemoglobin breakdown products are known to bind to the erythrocyte membrane [Goldberg and Stern (30)], and lipid peroxidation, which precedes hemolysis [Cohen (31)], is strongly stimulated by iron compounds [Tappel (32)]. Thus, the high correlation between hemoglobin breakdown and hemolysis observed in the present experiment, indicate that the primary role of vitamin E is to prevent the initiation of peroxidation within erythrocyte membranes by hemoglobin breakdown products. Prevention of this peroxidation effectively prevents subsequent hemolysis. This provides the basis for an explanation of the effects Cu and Se had on hemolysis and their interactions with vitamin E, in that Cu and Se both affected hemoglobin breakdown, independent of vitamin E status. Subsequent production of hemolysis by these breakdown products occurred only when cells were deficient in vitamin E. Adequate Se increased hemoglobin breakdown in cells incubated without glucose, and had no effect on hemoglobin breakdown or hemolysis in the presence of glucose. This contrasts with results reported previously by Rotruck et al. (16). The presence or absence of functional catalase activity appear to explain these differences. In the erythrocyte, catalase and glutathione peroxidase are probably equally effective in H202 removal [Ganther et al. (29)]. In the cell system of Rotruck et al. (16), catalase was inhibited by the addition of azide. Glutathione peroxidase then becomes the major mechanism for H202 removal, to prevent H202-induced oxidation of hemoglobin. Under these conditions, the requirement for glutathione peroxidase (and therefore Se) is greatly amplified, and the glucosedependent regeneration of GSH is of major importance. The present results
COPPER-SELENIUM-VITAMIN E INTERACTIONS
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indicate that this requirement for glutathione peroxidase is greatly diminished when cells contain normal activities of catalase. The increased hemoglobin breakdown in Se-adequate cells, when glucose is limiting, indicates that glutathione peroxidase may actually compete with catalase for H202 to the detriment of the cell. Reduction of glutathione by glutathione reductase requires NADPH [Paglia and Valentine (18)]. Methemoglobin reductase systems present in erythrocytes also require NADPH and NADH [Hultquist et al. (33)]. Thus, increased glutathione peroxidase activity, with its associated increased requirements for GSH regeneration, are largely replaceable in function by catalase, but will competitively inhibit methemoglobin reduction when glucose is limiting. Increased hemoglobin breakdown is the final result. Effects of dietary Cu also appeared to be mediated predominantly through hemoglobin metabolism. Cells depeleted of CuSOD had significantly reduced hemoglobin breakdown, and in some cases hemolysis, compared to cells with normal CuSOD activity. This contrasts with the reports by Goldberg and Stern (30) and Lynch et al. (34), that CuSOD prevents methemoglobin formation from hemoglobin. However, it confirms similar effects of CuSOD on hemolysis reported by Kellogg and Fridovich (3) and Michelson (35) for in vitro studies using mostly normal cells (in respect to CuSOD, glutathione peroxidase activities, and vitamin E status), with additional SOD and catalase added to the incubation medium. Kellogg and Fridovich (3) proposed that SOD had two effects in erythrocytes: the first was to block the O2 dependent conversion of methemoglobin to oxyhemoglobin. The second was to prevent the reaction of 02 with H202 to produce .OH (the Haber-Weiss reaction), thus preventing initiation of peroxidative damage and subsequent cell lysis by this latter radical species. In the present study, only the hemoglobin reactions were evident. 02 mediated perodixation in the cytoplasm, which should be inhibited by CuSOD and glutathione peroxidase, was not significantly involved in hemolysis; twentyfold differences in glutathione peroxidase activity (through dietary Se changes), even with adequate GSH, did not affect hemolysis in Cu-deficient cells. The results of this erythrocyte hemolysis experiment appear to question direct roles for both CuSOD and glutathione peroxidase in preventing peroxidation in erythrocytes. However, the erythrocyte because of its high heme content, and the dominant effects these enzymes have on hemoglobin metabolism under conditions of high oxidative stress, probably represents a special case in terms of peroxidative pathways. Further studies are required, using tissues other than the erythrocyte in which peroxidation mechanisms are not dominated by high concentrations of hemoglobin, before the theory of antioxidant functions of these enzymes in vivo can be discarded.
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Acknowledgments The a u t h o r gratefully acknowledges the capable technical assistance of Mr. B. S. Fleming and Mrs. S. Colgrave, and the many helpful discussions with Professors R. J. Moir and E. J. Underwood. This work was supported by funds provided by the Australian Wool Corporation, as part of a postgraduate scholarship held by D. I. Paynter.
References I. T. Noguchi, A. H. Cantor, and M. L. Scott, J. Nutr. 103, 1502 (1973). 2. W. G. Hoekstra, in Trace Element Metabolism inAnimals-2, W. G. Hoekstra, J. W. Suttie, H. E. Ganther, and W. Mertz, eds., University Park Press, Baltimore, 1974, pp. 61-77. 3. E. W. Kellogg and I. Fridovich, J. Biol. Chem. 252, 6721 (1977). 4. D. D. Tyler, FEBS Lett. 51, 180 (1975). 5. T. C. Pederson and S. D. Aust, Biochem. Biophys. Res. Commun. 52, 1071 (1973). 6. J. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). 7. R. A. Weisigner and I. Fridovich, J. Biol. Chem. 248, 3582 (1973). 8. D. I. Paynter, R. J. Moir, and E. J. Underwood, J. Nutr. 109, 1566 (1979). 9. D. G. Hafeman, R. A. Sunde, and W. G. Hoekstra, Z Nutr. 104, 580 (1974). 10. K. Schwarz, Fed. Proc. 24, 58 (1965). 11. L. S. Jensen, Proc. Soc. Expl. Biol. Med. 149, 113 (1975). 12. Hill, C. H., in Trace Elements in Human Health and Disease, vol. 2, A. S. Prasad, ed., Academic Press, New York, 1976, pp. 281-300. 13. M.K. Hill, S. D. Walker, and A. G. Taylor, N. Z. J. Agric. Res. 12,261 (1969). 14. G. G. Thomson and B. M. Lawson, N. Z. Vet. J. 18, 79 (1970). 15. M. L. Quaife and P. L. Harris, Ind. Eng. Chem. Anal Ed. 18, 707 (1946). 16. J. T. Rotruck, A. L. Pope, H. E. Ganther, and W. G. Hoekstra, J. Nutro 102, 689 (1972). 17. E. Beutler, O. Duron, and B. M. Kelly, J. Lab. Clin. Med. 61,882 (1963). 18. D. E. Paglia and W. N. Valentine, J. Lab. Clin. Med. 70, 158 (1967). 19. D. I. Paynter, Aust. J. Agric. Res. 30, 695 (1979). 20. G. B. Storer, Biochem. Med. 11, 71 (1974). 21. E. J. van Kampen and W. G. Zijlstra, Clin. Chim. Acta. 6, 538 (1961). 22. J. H. Watkinson, Anal Chem. 38, 92 (1966). 23. G. W. Snedecor and W. G. Cochran, in Statistical Methods, Iowa State University Press, Ames, Iowa, 1967, pp. 250-253, 359-361. 24. P. A. Abraham and J. L. Evans, in Trace Substances in EnvironmentalHealth, vol. 5, D. D. Hemphill, ed., University of Missouri, Columbia, 1972, pp. 335-347. 25. H. W. Bennetts, A. B. Beck, and R. Harley, Aust. Vet. J. 24, 237 (1948). 26. E.J. Underwood, in Trace Elements in Human and Animal Nutrition, 4th Ed., Academic Press, New York, 1977, pp. 84-86.
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27. J. R. Prohaska and H. E. Ganther, Biochem. Biophys. Res. Commun. 76, 437 (1977). 28. R. A. Lawrence and R. F. Burk, J. Nutr. 108, 211 (1978). 29. H. E. Ganther, D. G. Hafeman, R. A. Lawrence, R. E. Serfass, and W. G. Hoekstra, in Trace Elements in Human Health and Disease, vol. 2, A. S. Prasad, ed., Academic Press, New York, 1976, pp. 165-234. 30. B. Goldberg and A. Stern, Arch. Biochem. Biophys, 178, 218 (1977). 31. G. Cohen, in Erythrocyte Structure and Function, G. L. Brewer, ed., Liss, New York, 1975, pp. 685-698. 32. A. L. Tappel, Fed. Proc. 32, 1870 (1973). 33. D. E. Hulquist, R. H. Douglas, and R. T. Dean, in Erythrocyte Structure and Function, G. L. Brewer, ed., Liss, New York, 1975, pp. 297-301. 34. R. E. Lynch, G. R. Lee, and G. E. Cartwright, J. Biol. Chem. 251, 1015(1976). 35. A. M. Michelson, in Superoxide and Superoxide Dismutases, A. M. Michelson, J. M. McCord, and I. Fridovich, eds., Academic Press, New York, 1977, pp. 245-255.
Investigations into Combined Dietary Deficiencies of Copper, Selenium, and Vitamin E in the Rat D. I. PAYNTER*~" "'Attwood" Veterinary Research Laboratory, Mickleham Road, Westmeadows, Victoria, Australia, 3047 AND
G. B. MARTIN Department of Animal Science and Production, University of Western Australia, Nedlands, Western Australia, 6009 Received November 6, 1979; Accepted February 23, 1980
Abstract Interactions between dietary Cu, Se, and vitamin E in ascorbate-induced hemolysis of erythrocytes obtained from rats fed diets deficient or adequate in these elements were investigated. Hemolysis was affected by all three dietary factors, through closely interrelated but distinct mechanisms. In vitamin Edeficient cells, hemolysis was increased and the amount of hemolysis was directly related to the amount of hemoglobin breakdown. Deficiency of Cu or Se decreased hemolysis, but only in vitamin E-deficient cells. Vitamin E did not affect the breakdown of hemoglobin, but Cu and Se did. Hemolysis and hemoglobin breakdown were decreased by the addition of glucose, through mechanisms independent of that involving reduced glutathione metabolism. These results suggest that vitamin E acts within erythrocyte membranes to prevent products of hemoglobin breakdown from initiating peroxidation and subsequent hemolysis. Effects of Cu and Se are linked with that of vitamin E by the involvement of glutathione peroxidase and Cu superoxide dismutase in the cytoplasmic breakdown of hemoglobin, rather than by a direct effect of ~Previous address: Department of Animal Science and Production, University of Western Australia, Nedlands, Western Australia 6009. 9 1980 The Humana Press Inc. All rights of any nature whatsoever reserved. 0163 -498,t / 80 / 06004) 175$03.40
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these enzymes on lipid peroxidation. It is concluded that the erythrocyte, because of its high heine content, probably represents a special system in terms of peroxidative pathways, and these findings may not be directly applicable to other tissues. Index Entries: Copper, interactions with Se and Vitamin E in rats; selenium, interactions with Cu and vitamin E in rats; vitamin E, interactions with Cu and Se in rats; dietary deficiencies, of Cu, Se, and vitamin E in rats; interactions of Cu, Se, and vitamin E in rats; hemolysis mechanisms, and Cu-Se-vitamin E interactions in rats.
Introduction The role of selenium (Se) in glutathione peroxidase, and its interaction with vitamin E, has been postulated to operate through the role of these elements in peroxide metabolism [Noguchi et al. (I); Hoekstra, (2)]. Although the actual in vivo mechanisms leading to peroxide formation have not been fully elucidated, it appears that superoxide (O2)or its reaction products may play a significant role in peroxide initiation [Kellogg and Fridovich (3); Tyler (4); Peterson and Aust (5)]. Hence, one of the roles postulated for the superoxide dismutase enzymes is that of the removal of O~ as a primary defense against deleterious peroxidation. Two forms of superoxide dismutase have been characterized in mammalian cells; one form contains Cu and Zu (CuSOD) [McCord and Fridovich (6)], and is predominantly cytoplasmic, while the second form contains Mn and appears at least in some species to be localized in the mitochrondria [Weisigner and Fridovich (7)], In the rat, liver and erythrocyte CuSOD activity is rapidly depleted with dietary Cu deficiency [Paynter et. al. (8]. In the liver of the rat, activity of glutathione peroxidase, which also has a predominantly cytoplasmic localization, is rapidly depleted with dietary Se deficiency [Hafeman et al. (9)]. This tissue also appears to be most susceptible to a combined Se-vitamin E deficiency in this species, and under appropriate conditions, liver necrosis is characteristic of this deficiency [Schwarz (10)]. Hence, a combined deficiency of Cu, Se, and vitamin E could render tissues such as the liver particularly susceptible to peroxide damage, through both an increased amount of peroxide formation, and a decreased rate of removal of these peroxides. Interactions between Cu and Se in which the toxicity or availability of one of these elements is reduced by increasing the dietary concentration of the other, have been reported previously [Jensen (11); Hill (12)] The mechanisms of such interactions are at a different level from those interactions that can be proposed at more physiological, and in particular,
COPPER-SELENIUM-VITAMIN E INTERACTIONS
177
dietarily deficient concentrations of these elements, based on changes in apparently interacting enzyme systems. At these lower dietary concentrations, significant interactions between Cu and Se have been reported for production related parameters in sheep [Hill et al. (13); Thomson and Lawson (14)]. The possible mechanisms of these interactions have not been investigated. The aim of the experiments to be described was to determine the effects of Cu deficiency in rats deficient in Se and/or vitamin E. Emphasis was placed on the biochemical changes relating to these deficiencies, and the use of the erythrocyte as an in vitro model system, in an attempt to determine more fully the physiological significance of these dietarily-induced changes.
Materials and Methods Animals and Diets Twenty-one day old weanling male rats of the Sprague-Dawley strain were used in the experiments. These rats were derived from females fed a Sedeficient diet (as shown in Table 1, supplemented with Cu and vitamin E) from the time of mating onwards. At weaning, rats were housed individually in stainless steel and plastic cages at constant temperature (25~ and controlled lighting (lighting 0600 to 1900 hours), and feeding of the experimental diets was begun. The composition of the basal diet is shown in Table 1. Each of the three dietary variables (Cu, Se, and vitamin E) was used at two concentrations; deficient (-), and adequate (+), giving a total of eight dietary combinations. Deficient concentrations were those present in the basal diet, i.e., 0.5 mg Cu/kg and 0.025 mg Se/kg. Vitamin E content was not assayed directly, but was estimated to be less than 2 mg/kg, based on the reported vitamin E content of lard [Quaife and Harris (15)]. For the adequate diets, Cu was added (as CuSO4 9 5H20) at 10 mg Cu/kg diets, Se (as Na2SeO3) at 0.25 mg Se/kg and vitamin E (as dl-a-tocopherol acetate) at 100 mg/kg diet. Each dietary group contained 10 rats. Feed and deionized water were supplied ad libitum to all rats throughout the experiment. Feed residues were collected and weighed daily and rats were weighed weekly. Tissue Sampling Tissue samples for hemolysis and enzyme activity studies were obtained from rats that had been fed the diets for 50 days. These rats were randomly selected from those remaining in each dietary group at this time.Following light etherization, blood was obtained by cardiac puncture using heparinized syringes, and quickly chilled in ice water. Required organs were removed, washed in cold 154 mM NaC1, then placed in aluminum foil containers chilled on cracked ice.
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TABLE 1 Composition of the Basal Diet Ingredient Caseina Glucose monohydrate Salt mixb Vitamin mixc Lard Cod liver oil DL-methionine Choline chloride
g/kg diet 200 660 53.4 2.5 60 20 4 0.522
aSunnywest Diaries, Perth, Western Australia. bProvided the followingas g/kg diet: NaH2PO~.2H20, 26.0; CaCO3, 18.2; KCI,3.5; Na2CO~, 1.2;MgSO4.7H~O, 3.99; MnSO4.H20, 0.15; FeSO4'7H20, 0.249; ZnSO4.7H~O,0.132; NaF, 0.00025; (NH4)6MoTO24"4H20, 0.002; CoSO4.THzO,0.01; KI, 0.0003. CProvided the followingas mg/kg diet: thiamin-HCl, 12; niacin, 100; inositol, 100;p-aminobenzoicacid, 100; Ca pantothenate, 30; riboflavin, 15; pyridoxine-HCl, 12; folic acid, 2; menadione,1.5;biotin,0.5; cyanocobalamin, 0.03; retinyl palmitate (5 X 105 USP units/g), 6; ergocalciferol (4 X 107 USP units/g), 0.05.
Erythrocyte Hemolysis Studies The method used for the erythrocyte hemolysis study was essentially that described by Rotruck et al. (16), with some modifications. From each animal's whole blood, duplicate samples were taken each containing the equivalent of 0.30 mL packed cells (calculated from the hematocrit value for the whole blood), and added to tubes containing either 10 mL of 152 m M NaC1 + 10mM NazHPO4, pH 7.4 (PBS), or 10 mL of PBS + 11 m M glucose. After mixing and centrifugation, the supernatants were removed by aspiration, and a further 10 mL of PBS or PBS + glucose was added to the respective tubes. Ascorbate (0.1 mL of 220 m M Na ascorbate in PBS, pH 7.4) was then added and the tubes incubated at 37~ Until this incubation step, all operations were performed at 4~ For hemolysis determination, 0.5 mL samples of incubating cells were withdrawn, and added to tubes containing either 3 mL of PBS or 3 mL of water only. Following centrifugation, absorbance at the supernatant solutions was measured at 540 nm. From these absorbances, hemolysis and hemoglobin breakdown (decrease in absorbance of 540 nm, of totally hemolyzed sample, using To value as reference) were estimated. Reduced glutathione (GSH) was measured (as sulfhydryl) by the method of Beutler et al. (17) with modifications as described by Rotruck et al. (16). Samples for
COPPER-SELENIUM-VITAMIN E INTERACTIONS
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the estimation of hemolysis, hemoglobin breakdown, and GSH concentrations, were taken at 0, 1, 3, and 6 h after beginning the ascorbate incubations at 37~
Enzyme and Other Assays For measurement of glutathione peroxidase and CuSOD in heart and liver, 10% (W/V) homogenates of these tissues were prepared in 0.2% (V/V) Triton-X100, using glass-glass tissue homogenizers. For measuring activities in erythrocytes, whole blood was first centrifuged and the plasma removed. The cells were then washed three times in PBS and after the final wash, a sample of the packed cells was diluted 1 in 5 with water. Glutathione peroxidase activity was measured using the method of Paglia and Valentine (18), with modifications as described previously by Paynter (19). One unit of activity (U) is equivalent to 1 #mol of NADPH oxidized per min at 25~ CuSOD activity in chloroform-ethanol treated homogenates and lysates, was determined by the method of McCord and Fridovich (6), with minor modifications [Paynter et al. (8)]. Units of activity are as described by McCord and Fridovich (6). Vitamin E concentration in plasma was determined by the fluorimetric method of Storer (20). Plasma asparate aminotransferase activity was measured using a commercial test kit method (Boehringer Mannheim). Hemoglobin content of whole blood was determined by the cyanomethemoglobin method [Kampen and Zijlstra (21)]. Concentration of Se in diets was determined fluorimetrically [Watkinson (22)]. Cu was measured using conventional flame atomic absorption spectometry, following wet ashing of samples with nitric-perchloric acids.
Statistics Chi-square analysis was used for testing the significance of treatments on the incidence of rat deaths [Snedecor and Cochran (23)]. With all other data, differences between treatments were tested for significance using a factorial analysis [Snedecor and Cochran (23)]. Values for glutathione peroxidase activities and plasma vitamin E concentrations were logt0 transformed before analysis. Arcsine transformations were applied to the erythrocyte hemolysis data, with glucose included as an additional factor in this analysis.
Results Rats began dying after the experimental diets had been fed for 5 weeks. At this time, Cu was the only dietary factor that significantly affected bodyweight (Table 2). By 7 weeks, the increasing frequency of deaths in
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some groups, forced the termination of the experiment. All deaths were confined to the - C u diets and although more rats died when fed - C u - S e compared to - C u +Se diets (9/20 compared to 3/20) only the effects of Cu were significant. In all rats that died, post-mortem examination revealed large amounts of clotted blood in the chest cavity owing to rupture of the heart in the region of the ventricular apex. Heart ventricles were grossly enlarged with extensive fibrosis evident at the apex. In dead rats heart weights were increased up to 3-fold compared to rats of similar bodyweight fed adequate diets. In the rats that were killed at 7 weeks, only Cu significantly affected heart weights. Cu deficiency also resulted in small but significant decreases in blood hemoglobin and packed cell volumes after feeding the diets for 7 weeks. No significant effects (or interactions) of either Se or vitamin E were observed in these parameters (Table 2). In contrast, aspartate aminotransferase activities in plasma were increased in rats fed - S e and -vitamin E diets, and there were significant vitamin E X Se and Se • Cu interactions (Table 3). Activities of CuSOD and glutathione peroxidase in erythrocytes, liver, and heart tissues of rats fed the various diets for 7 weeks are shown in Table 3. In rats fed - S e diets, glutathione peroxidase activities were reduced to less than 5% that in +Se rats for all tissues assayed. Cu deficiency decreased CuSOD activity in liver, erythrocyte, and heart to 17, 25, and 43%, respectively, of control activities. Cu deficiency increased glutathione peroxidase activities in the erythrocyte and to a lesser extent in the liver; plasma vitamin E and erythrocyte GSH concentrations were both increased (Table 3; Fig. 1). In the in vitro hemolysis studies, all dietary factors significantly affected hemolysis, but the major dietary effect was associated with vitamin E. Effects of dietary Cu and Se were only observed with erythrocytes from vitamin E-deficient animals (Fig. 1A, B). Inclusion of glucose decreased the amount of hemolysis in-vitamin E erythrocytes, but did not affect the small amount of hemolysis in +vitamin E cells. Compared to the effects of vitamin E on hemolysis, the reverse was observed with dietary Se in vitamin Edeficient cells. Without glucose, -Se-vitamin E cells had less hemolysis than +Se -vitamin E cells. With added glucose this effect disappeared, and Se status of the erythrocytes did not affect hemolysis regardless of the vitamin E or Cu status of these cells. Similar effects of dietary Se on hemolysis were also observed for Cu, with cells from - C u -vitamin E rats hemolyzing less than cells from +Cu -vitamin E rats. However, in contrast to dietary Se, where effects were only observed in the absence of glucose, these effects of Cu were observed both in the presence and absence of glucose in the incubation mixture (Figs. 1A, B). Inhibition of hemolysis by glucose was in general associated with a maintenance of erythrocyte GSH concentrations (Fig. 1C, D). However, maintenance of GSH did not prevent hemolysis as evidenced by the considerable hemolysis in erythrocytes from -vitamin E +Cu rats,
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COPPER-SELENIUM-VITAMIN E INTERACTIONS
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irrespective of Se and GSH status, when incubated in the presence of glucose (Fig. 1D). Without glucose in the incubation medium, there was a rapid decrease in erythrocyte GSH concentration with all cells in the first hour of incubation (Fig. 1C). These decreases continued until at least 3 h, after which GSH concentrations were generally increased. GSH increased the most in those - v i t a m i n E erythrocytes that were most intensively hemolyzed. The decreases in GSH concentration measured in the first hour of incubation without glucose were significantly greater in cells from - C u rats. These cells were also higher in GSH compared to other cells, prior to incubation (Fig. 1C, D). Neither vitamin E nor Se status of the cells significantly affected GSH concentrations, either before or in the first hour of incubation with ascorbate. As in the cells incubated with added glucose, the GSH content of cells was not related to the degree of hemolysis. Relative breakdown of hemoglobin in cells incubated without glucose for 6 h was significantly affected by dietary selenium only. In the presence of glucose, overall hemoglobin breakdown was decreased, and only dietary Cu significantly affected this breakdown. In both cases where significant dietary effects were obtained, cells from supplemented rats, i.e., +Se or +Cu, showed greater hemoglobin breakdown than cells from deficient rats (Fig. 2). Dietary vitamin E had no effect on hemoglobin breakdown. However, the amount of hemoglobin breakdown was highly correlated to the amount of hemolysis in -vitamin E erythrocytes at 6 h (Fig. 3). In these cells, a continuous correlation was apparent between hemoglobin breakdown and hemolysis. The continuous nature of this relationship was not affected by glucose status of cells or by dietary effects of Cu and Se. In +vitamin E cells, this correlation was not present, minimal hemolysis occurred in the presence of considerable hemoglobin breakdown.
Discussion In contrast to previously reported interactions between Cu and Se in the growth of sheep [Hill et al. (13); Thomson and Lawson (14)], only Cu significantly affected growth in the present experiment. At the tissue level, deficiency of Cu was associated with hypertrophy and fibrosis of the heart. The incidence of fatal heart lesions in these rats was increased 3-fold with a concurrent Se deficiency. Although hypertrophy of the heart is a consistent feature of Cu deficiency in the rat [Abraham and Evans (24)], extensive fibrosis occurring at the apex is not. Venticular fibrosis, similar to that observed in this experiment, has been described by Bennetts et al. (25) with "falling disease" in cattle, a disease responsive to Cu, but not always associated with even severe Cu deficiency [Underwood (26)]. The present results indicate that although a dominant role is played by Cu in the development of these lesions, other factors, including selenium, may also be involved.
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FIG. 3. Relationship between hemoglobin breakdown and hemolysis for erythrocytes, after 6 h incubation in the presence of ascorbate. Values shown were those obtained for individual rats, fed diets containing_+ Se and _+ Cu. Erythrocytes from +vitamin E rats, incubated with (D), and without glucose (9 Erythrocytes from -vitamin E rats, incubated with ( . ) , and without glucose (e). Liver necrosis was not observed in any of the dietary treatments even though changes in glutathione peroxidase and CuSOD activities were greatest in this tissue. This indicates that factors additional to these enzymes are involved in determining gross organ damage. These additional factors include other enzymes requiring the same dietary elements and other enzymes able to catalyze the same reactions, but not dependent on the same dietary elements for activity. An example of the former is the variable tissue effect of Cu deficiency on cytochrome oxidase activity compared with CuSOD [Paynter et al. (8)]. Examples of the latter include the manganesecontaining SOD that may compensate for changes in CuSOD activity, and the non-Se-dependent glutathione peroxidase, i.e., the glutathione transferases [Prohaska and Ganther (27)] compensating for the Sedependent enzyme. In erythrocytes, many of these complications are either minimal or absent. Thus, glutathione transferase activity is not present in these cells [ Lawrence and Burk (28)] and they do not contain mitochondrial-associated enzymes such as cytochrome oxidase. In the present experiment, several
188
PAYNTER AND MARTIN
apparently interacting effects were observed between Cu and Se in erythrocytes. The major effect was the increases in both measured glutathione peroxidase activity and in GSH concentrations with Cu deficiency. Since the reaction rate of glutathione peroxidase with peroxide substrates is dependent on GSH concentration [Ganther et al. (29)], the potential activity of this enzyme will be significantly increased with Cu deficiency. Small increases in plasma vitamin E were also associated with Cu deficiency in the present experiment. These changes, taken together, may indicate that compensating mechanisms are operating in vivo for the decreased CuSOD activity in Cu-deficient erythrocytes. This concept was not supported by the results of the in vitro hemolysis experiment. The results of this experiment were also unexpected, based on previously postulated antioxidant functions of glutathione peroxidase and SOD. Although hemolysis increased with vitamin E deficiency, Cu and Se deficiencies both decreased hemolysis. These effects of Cu and Se were confined to vitamin E-deficient cells, in which the amount of hemolysis directly related to the amount of hemoglobin breakdown. Hemoglobin breakdown products are known to bind to the erythrocyte membrane [Goldberg and Stern (30)], and lipid peroxidation, which precedes hemolysis [Cohen (31)], is strongly stimulated by iron compounds [Tappel (32)]. Thus, the high correlation between hemoglobin breakdown and hemolysis observed in the present experiment, indicate that the primary role of vitamin E is to prevent the initiation of peroxidation within erythrocyte membranes by hemoglobin breakdown products. Prevention of this peroxidation effectively prevents subsequent hemolysis. This provides the basis for an explanation of the effects Cu and Se had on hemolysis and their interactions with vitamin E, in that Cu and Se both affected hemoglobin breakdown, independent of vitamin E status. Subsequent production of hemolysis by these breakdown products occurred only when cells were deficient in vitamin E. Adequate Se increased hemoglobin breakdown in cells incubated without glucose, and had no effect on hemoglobin breakdown or hemolysis in the presence of glucose. This contrasts with results reported previously by Rotruck et al. (16). The presence or absence of functional catalase activity appear to explain these differences. In the erythrocyte, catalase and glutathione peroxidase are probably equally effective in H202 removal [Ganther et al. (29)]. In the cell system of Rotruck et al. (16), catalase was inhibited by the addition of azide. Glutathione peroxidase then becomes the major mechanism for H202 removal, to prevent H202-induced oxidation of hemoglobin. Under these conditions, the requirement for glutathione peroxidase (and therefore Se) is greatly amplified, and the glucosedependent regeneration of GSH is of major importance. The present results
COPPER-SELENIUM-VITAMIN E INTERACTIONS
189
indicate that this requirement for glutathione peroxidase is greatly diminished when cells contain normal activities of catalase. The increased hemoglobin breakdown in Se-adequate cells, when glucose is limiting, indicates that glutathione peroxidase may actually compete with catalase for H202 to the detriment of the cell. Reduction of glutathione by glutathione reductase requires NADPH [Paglia and Valentine (18)]. Methemoglobin reductase systems present in erythrocytes also require NADPH and NADH [Hultquist et al. (33)]. Thus, increased glutathione peroxidase activity, with its associated increased requirements for GSH regeneration, are largely replaceable in function by catalase, but will competitively inhibit methemoglobin reduction when glucose is limiting. Increased hemoglobin breakdown is the final result. Effects of dietary Cu also appeared to be mediated predominantly through hemoglobin metabolism. Cells depeleted of CuSOD had significantly reduced hemoglobin breakdown, and in some cases hemolysis, compared to cells with normal CuSOD activity. This contrasts with the reports by Goldberg and Stern (30) and Lynch et al. (34), that CuSOD prevents methemoglobin formation from hemoglobin. However, it confirms similar effects of CuSOD on hemolysis reported by Kellogg and Fridovich (3) and Michelson (35) for in vitro studies using mostly normal cells (in respect to CuSOD, glutathione peroxidase activities, and vitamin E status), with additional SOD and catalase added to the incubation medium. Kellogg and Fridovich (3) proposed that SOD had two effects in erythrocytes: the first was to block the O2 dependent conversion of methemoglobin to oxyhemoglobin. The second was to prevent the reaction of 02 with H202 to produce .OH (the Haber-Weiss reaction), thus preventing initiation of peroxidative damage and subsequent cell lysis by this latter radical species. In the present study, only the hemoglobin reactions were evident. 02 mediated perodixation in the cytoplasm, which should be inhibited by CuSOD and glutathione peroxidase, was not significantly involved in hemolysis; twentyfold differences in glutathione peroxidase activity (through dietary Se changes), even with adequate GSH, did not affect hemolysis in Cu-deficient cells. The results of this erythrocyte hemolysis experiment appear to question direct roles for both CuSOD and glutathione peroxidase in preventing peroxidation in erythrocytes. However, the erythrocyte because of its high heme content, and the dominant effects these enzymes have on hemoglobin metabolism under conditions of high oxidative stress, probably represents a special case in terms of peroxidative pathways. Further studies are required, using tissues other than the erythrocyte in which peroxidation mechanisms are not dominated by high concentrations of hemoglobin, before the theory of antioxidant functions of these enzymes in vivo can be discarded.
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Acknowledgments The a u t h o r gratefully acknowledges the capable technical assistance of Mr. B. S. Fleming and Mrs. S. Colgrave, and the many helpful discussions with Professors R. J. Moir and E. J. Underwood. This work was supported by funds provided by the Australian Wool Corporation, as part of a postgraduate scholarship held by D. I. Paynter.
References I. T. Noguchi, A. H. Cantor, and M. L. Scott, J. Nutr. 103, 1502 (1973). 2. W. G. Hoekstra, in Trace Element Metabolism inAnimals-2, W. G. Hoekstra, J. W. Suttie, H. E. Ganther, and W. Mertz, eds., University Park Press, Baltimore, 1974, pp. 61-77. 3. E. W. Kellogg and I. Fridovich, J. Biol. Chem. 252, 6721 (1977). 4. D. D. Tyler, FEBS Lett. 51, 180 (1975). 5. T. C. Pederson and S. D. Aust, Biochem. Biophys. Res. Commun. 52, 1071 (1973). 6. J. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). 7. R. A. Weisigner and I. Fridovich, J. Biol. Chem. 248, 3582 (1973). 8. D. I. Paynter, R. J. Moir, and E. J. Underwood, J. Nutr. 109, 1566 (1979). 9. D. G. Hafeman, R. A. Sunde, and W. G. Hoekstra, Z Nutr. 104, 580 (1974). 10. K. Schwarz, Fed. Proc. 24, 58 (1965). 11. L. S. Jensen, Proc. Soc. Expl. Biol. Med. 149, 113 (1975). 12. Hill, C. H., in Trace Elements in Human Health and Disease, vol. 2, A. S. Prasad, ed., Academic Press, New York, 1976, pp. 281-300. 13. M.K. Hill, S. D. Walker, and A. G. Taylor, N. Z. J. Agric. Res. 12,261 (1969). 14. G. G. Thomson and B. M. Lawson, N. Z. Vet. J. 18, 79 (1970). 15. M. L. Quaife and P. L. Harris, Ind. Eng. Chem. Anal Ed. 18, 707 (1946). 16. J. T. Rotruck, A. L. Pope, H. E. Ganther, and W. G. Hoekstra, J. Nutro 102, 689 (1972). 17. E. Beutler, O. Duron, and B. M. Kelly, J. Lab. Clin. Med. 61,882 (1963). 18. D. E. Paglia and W. N. Valentine, J. Lab. Clin. Med. 70, 158 (1967). 19. D. I. Paynter, Aust. J. Agric. Res. 30, 695 (1979). 20. G. B. Storer, Biochem. Med. 11, 71 (1974). 21. E. J. van Kampen and W. G. Zijlstra, Clin. Chim. Acta. 6, 538 (1961). 22. J. H. Watkinson, Anal Chem. 38, 92 (1966). 23. G. W. Snedecor and W. G. Cochran, in Statistical Methods, Iowa State University Press, Ames, Iowa, 1967, pp. 250-253, 359-361. 24. P. A. Abraham and J. L. Evans, in Trace Substances in EnvironmentalHealth, vol. 5, D. D. Hemphill, ed., University of Missouri, Columbia, 1972, pp. 335-347. 25. H. W. Bennetts, A. B. Beck, and R. Harley, Aust. Vet. J. 24, 237 (1948). 26. E.J. Underwood, in Trace Elements in Human and Animal Nutrition, 4th Ed., Academic Press, New York, 1977, pp. 84-86.
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