Effect of copper sulfate on experimental atherosclerosis

�9 1993 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/93/3801-0047 $03.00

Effect of Copper Sulfate on Experimental Atherosclerosis

MARIANA VLAD, 1 E. BORDAS, 1 RODICA TOMUS,' DOINA SAVA, ~

ECATERINA FARKAS, ~ AND G. Uz_A 2,*

1Institute of Public Health and Medical Research and 2Departmental Policlinic 11, Cluj-Napoca, Romania

Received March 1, 1992; Revised October 25, 1992; Accepted November 23, 1992

ABSTRACT

Serum copper concentration increases significantly (p < 0.01) in rats with experimental atherosclerosis compared to a control group. The serum zinc, the zinc, and copper concentration in abdominal aorta and in liver decreases significantly (p < 0.05) compared to the control group. Administration of copper sulfate for 100 d in these animals induces a significant increase of serum copper (p < 0.01), decrease of serum cholesterol (p < 0.05) and increase of liver copper concentration as compared with the group fed only a high cholesterol diet. In the aorta of these animals the copper concentration increases and edema and lipid infiltration are considerably less than in the group of animals fed only a high lipid diet.

Index Entries: Serum Cu and Zn; tissue Cu and Zn concentration; copper sulfate; experimental atherosclerosis.

INTRODUCTION

Copper-deficient rats have been found with an increased cholesterinemia and increased LDL-cholesterol together with a decrease of HDL-cholesterol (1-6). Other authors showed that the increase of serum cholesterol is associated with the increase of HDL-cholesterol (3,4,7).

*Author to whom all correspondence and reprint requests should be addressed.

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48 Wad et aL

Hypercholester inemia was also reported in humans with copper dietetic deficit (8,9). Starting from these data it was supposed that rela- tive or absolute Cu deficit, characterized by an increase of Zn/Cu ratio could be a risk factor in the etiology of coronary heart disease (1,2). It was recently found that in patients with hyperl ipoproteinemia there is a decrease of serum Cu and Zn/Cu ratio (10,11). The decrease of serum Zn concentration in these patients does not seem to be connected with serum lipides, but rather with age (11).

In this paper we have examined the effect of copper sulfate on the concentrat ion of serum cholesterol and the serum and tissue Cu and Zn in rats fed a cholesterol rich diet.

MATERIAL AND METHODS

Experimental ]Vlaterial

The experiments were performed on albino male Wistar rats, with an initial mean body wt of 167 ___ 29 g kept in vivarium conditions and fed a s tandard diet containing 0.13 mg Cu/animal/d and 0.01 mg cholesterol/ animal/d.

The experimental animals were divided into three groups:

Group A: Controls receiving standard diet (30 animals)

Group B: Animals fed a diet supplemented with cholesterol (Serva-Heidelberg) given in a dose of 40 mg/animal/d p.o. for a period of 60 d in 50% of animals and in 50% for 100 d (30 animals)

Group C: Animals fed with a cholesterol-supplemented diet and treated with 20 mg/animal/d p.o. copper sulfate in 1 mL 9%. NaC1 given through a gastric tube (30 animals).

After 60 d, 50% of the animals were sacrificed. The remaining animals were killed after 100 d. After sacrifice the abdominal aorta, liver, and kidneys were examined. The sections performed in series were stained with hematoxilin-eosin, Van Giesen, and Tricrom.

Serum Cholesterol and Minerals Analysis

The total, free, and esterified serum cholesterol was measured by the Assous and Girard methods (12).

The concentrations of Cu and Zn in the serum and organs were de termined at the beginning of the experiment, at 60 and 100 d, by means of a model 300 Perkin-Elmer atomic absorption spectrophotome- ter with flame atomization. The serum was measured with an automatic micropipet (500 bLL), and placed in metal-free Sovirel test tubes with teflon-stopper. The serum was diluted with bidistilled water in propor- tion of 1:1 for Cu and 1:2 for Zn.

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Experimental Atherosclerosis 49

Tissue determination of Cu and Zn was performed after a previous wet ashing, I g tissue was weighed in a metal-free Erlenmayer flask, then dried in a drying oven at 105~ The dried substance was weighed again, 10 mL concentrated HNO3 (d-1.4 Merck) was added, and covered with a watch glass. After 24 h it was warmed on a sandbath until the acid was evaporated, then 5 mL concentrated HNO3 and 1 mL of concentrated HCIO 4 were added. The fluid was evaporated on the sandbath until a dry, white-colored residue was obtained. The sample was cooled and added to 1 mL 1% HNO3.

Statistical Analysis

Statistical significance of the difference between the mean values was calculated by Student's t-test.

RESULTS

Serum Level of Cu, Zn, and Cholesterol

In the animals of group A the mean values of Cu, Zn, total, free, and esterified cholesterol in serum were not significantly changed during the experiment (Tables 1 and 2).

In the animals of group B, 60 d after beginning the experiment, serum Cu and cholesterol were increased but not significantly in comparison with the control group (Tables 1 and 2). After 100 d both significantly increased: cholesterinemia and serum Cu and Zn/Cu ratio significantly decreased (Table 1 and 2), compared to the values found in group A and to those recorded before treatment. At the same time the concentration of serum Zn decreased significantly compared with the concentration found in group A (Table 1).

In the animals of group C, 60 d after beginning the experiment, the level of serum Cu increased significantly and cholesterinemia was not significantly changed in comparison with the values found in groups A and B (Tables 1 and 2). After 100 d, serum Cu remained significantly increased in these animals compared with the values obtained in controls and very close to those recorded in group B (Table 1). Cholesterinemia is significantly decreased compared with the values found in group B (Table 2).

7issue Level of Cu and Zn

In the animals of group A the Cu concentration in abdominal aorta, liver, and kidneys was not significantly changed in comparison with the values found at the beginning of the experiment (Table 3).

In the animals of group B, the Cu and Zn concentration in abdominal aorta decreased significantly after 60 and 100 d of the experiment, compared to the control group (Table 3), but the Zn/Cu ratio did not change

Biological Trace Element Research Vol. 38, 1993

50 Wad etal.

Table 1 Se rum Cu a nd Z n Concent ra t ion (p,g/100 mL, Mean Values + SD) in Control Group (A),

the Group of An i ma l s Fed a Choles te ro l -Supp lemented Diet (B), and the Group Fed a Cho les te ro l -Supp lemented Diet and Treated With Coppe r Sulfate (C)

N Cu p,g % Zn gg % Zn/Cu

After After After After After After Initial 60 d 100 d Initial 60 d 100 d Initial 60 d 100 d

Group 121.2 120.5 119.9 108.6 108.9 109.7 0.90 0.91 0.91 A +6.21 _+5.32 • +6.82 +7.13 +4.25 --_0.07 +0.06 +0.04 (30)

Group 122.8 - 132.1 *'152.6 111.5 102.3 *'82.1 0.91 0.77 **0.54 B _+3.41 • +4 .8 _+2.04 _+4.12 +4.48 _+0.03 +0.05 +0.03 (30)

Group 120.4 *'150.8 *'152.8 109.1 105.9 103.3 0.91 *0.70 ~ 0.67 C _+4,63 +4.05 _+4.33 _+8.24 -+3.12 +8.21 --0.07 +0.05 _+0.06 (30)

Statistical significance: group B vs A: *p < 0.05 group C vs A: **p < 0.01 group B vs C: -p < 0.05

Table 2 Total, Free, and Esterified Se rum Cholesterol (mg/100mL, Mean Values -+ SD) in Control Group

(A), the Group Fed a Choles te ro l -Supplemented Diet (B), and the Group Fed a Cholesterol- S u p p l e m e n t e d Diet and Treated wi th Copper Sulfate (C)

N

Total s e r u m Esterified s e r u m Free s e r u m cholesterol cholesterol cholesterol mg/100mL mg/100mL mg/100mL

After After After After After After 60 d 100 d Initial 60 d 100 d Initial 60 d 100 d Initial

Group 61.0 66.0 68.2 36.3 41.8 43.3 24.7 24.2 25.0 A +2 .9 _+4.2 • _+3.3 +2 .0 +3 .7 -+2.3 _+3.5 +4 .6 (3O)

Group 60.2 76.9 - ~'91.9 35.2 53.4 **66.7 25.0 23.5 24.8 B _+7.2 +5 .4 • +3 .4 -+4.2 -+2.7 -+2.7 _+3.1 _+3.7 (30)

Group 61.3 71.7 76.6 36.0 48.8 55.1 25.3 22.3 21.5 C -+6.1 _+7.9 • _+2.1 +2 .8 +3.9 23 .4 _+3.6 +2.1 (30)

Statistical significance: group B vs A: *p < 0.05 group C vs A: **p < 0.01 group B vs C: -p < 0.05

significantly (Table 3). After 100 d, the concentration of liver Cu decreased significantly compared to groups A and C (Table 3). The concentration of Cu in kidneys was also decreased but not significantly in comparison with the Cu concentration found in groups A and C (Table 3). The level of Zn in liver and kidneys increased but not significantly (Table 3).

In the animals of group C, under the influence of the treatment with copper sulfate, administered for 60 d, the Cu concentration in liver and kidneys increased significantly compared with the concentrations found in groups A and B (Table 3). At the end of the experiment, after 100 d of treatment with copper sulfate, the Cu concentration in liver and kidneys


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52 Vlad et aL

Fig. 1. Abdominal aorta, rat, control group (A), with normal aspect.

was nearly identical with that found in the control group (Table 3). The differences in Zn values in liver and kidneys of groups B and C were not significant (Table 3).

JViorphological Changes Compared to the normal aspect of the control group, (Fig. 1), after 60

d of high lipid diet, animals of group B showed some edema and lipid infiltration of the intima. After 100 d edema and lipid infiltration were more evident, with some fragmentation of elastic fibers and thickening of the collagen fibers (Fig. 2).

In the animals of group C, after 60 d of treatment with copper sulfate and high lipid diet, the morphological changes in the aorta were considerably less than in the aorta of the animals of group B. After 100 d, minimal lipid sediments were observed (Fig. 3).

DISCUSSION

In rats with cholesterol supplement in the diet, within 60 d from the beginning of the experiment, the humoral and tissue changes were not significant. After 100 d the high lipid diet led to typical a theromatous changes. At the same time, a significant increases of serum Cu and decrease of serum Zn concentration were observed. The decrease of the Cu concentration in the aorta, liver, and kidneys in animals fed a high lipid diet may be a result of increased Cu transport to the extracellular space.

Decrease of Cu concentration in the liver has also been reported in humans dying with severe atherosclerotic lesions (13). Other authors

Biological Trace Element Research VoL 38, 199.3

Experimental Atherosclerosis 53

Fig. 2. Abdominal aorta, rat, fed with a diet supplemented with cholesterol. After 100 days from the beginning of the experience infiltration and edema of the intima were observed, to which are added fragmentation of elastic fibers and thickening of the collagen fibers.

Fig. 3. Abdominal aorta, rat, fed cholesterol supplement and treated with CuSO4. After 100 days, the morphological changes in the aorta were much more dimin- ished and minimal lipid sediments were revealed.

found small Cu concentrations in the atherosclerotic tissue (14,15). Con- sequently there is no correlation be tween the blood- and tissue-Cu concentrations. It has been suggested that reduced Cu in tissues makes the cells more avid for cholesterol (16).


54 Vlad et aL

In g r oup C, adminis t ra t ion of Cu sulfate reduces cholesterinemia. Se rum Cu level and aortic Cu concentrat ion also increase. In liver and k idneys the Cu concentra t ion increased only after 60 d of t rea tment wi th copper sulfate. After 100 d the values were nearly the same as in the control group. It is probable that dur ing this t ime the Cu shifted to the arterial tissues. As a consequence, the lipid infiltration in the arterial wall decreased.

In conclusion, in experimental atherosclerosis the concentrat ion of Cu in the abdominal aorta and the liver decreases significantly. The adminis t ra t ion of copper sulfate in these animals for 100 d induced a significant increase of se rum Cu level, aortic and liver Cu concentrat ion, a nd reduces se rum cholesterol concentrat ion in compar i son with the g roup of animals fed a cholesterol-rich diet. In the aorta of animals t reated wi th copper sulfate the edema and lipid infiltration are less than in the g roup fed only a h igh lipid diet.

REFERENCE8

1. M. L. Klevay, Am. J. Nutr. 26, 1060-1068 (1973). 2. K. G. D. Allen and L. M. Klevay, Atherosclerosis 31, 259-299 (1978). 3. K. Y. Lei, C. A. Hassel, and D. K. Allen, J. Nutr. 113, 2178-2183 (1983). 4. K. G. D. Allen and L. M. Klevay, Nutr. Rep. Int. 22, 295-299 (1980). 5. C. A. Hassel, T. P. Carr, J. A. Marchetlo, and K. Y. Lei, Proc. Soc. Exp. Biol.

Med. 187, 296-308 (1988). 6. P. W. Harvey and K. G. D. Allen, Nutr. Res. 5, 5ll-525 (1989). 7. T. P. Carr and K. Y. Lei, PSEBM 191, 370-376 (1989). 8. M. L. Klevay, L. Inman, L. K. Johnson, M. Lawler, J. R. Mahalko, D. B.

Milne, H. C. Lukaski, W. Bolonchuk, and H. H. Sandstead, Metabolism 33, 1112-1118 (1984).

9. S. Reiser, A. Powell, C. Yang, and J. J. Canary, Nutr. Rep. Int. 36, 641-649 (1987).

10. Y. Thuillier-Juteau, M. C. Jaudon, J. P. Clavel, J. Delattre, and A. Galli, Pathol. Biol. 35, 387-390 (1987).

11. G. Uza and R. Vlaicu, Biol. Tr. Elem. Res. 20, 197-206 (1989). 12. M. E. S. Assous and M. L. Girard, in Mitrica-Kondi Natalia, Laboratorul

Clinic, Biochimie, Ed. Med. Bucuresti, 1981, p. 210. 13. A. N. Howard, G. A. Greshak, and R. Sham, J. Tr. Elem. Exp. Med. 2,

134 (1989). 14. R. Masironi, in Hardness of drinking water and public health (Commission of

European Communities), Pergamon, Oxford, 1976, pp. 411 4~0. 15. D. Meissner, in Mengen und Spurenelemente, Leipzig, 198I, pp. 141-148. 16. T. C. Carr and K. Y. Lei, Metabolism 39, 518-524 (1990).


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Effect of copper sulfate on experimental atherosclerosis