0163-4984/93/3801-0047 $03.00. Effect of Copper Sulfate on Experimental Atherosclerosis. MARIANA VLAD, 1 E. BORDAS, 1 RODICA TOMUS,' DOINA SAVA,.
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_A2,*
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. Biological Trace Element Research
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Hypercholesterinemia was also reported in h u m a n s with copper dietetic deficit (8,9). Starting from these data it was supposed that relative 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 hyperlipoproteinemia 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 concentration of s e r u m 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 m e a n body wt of 167 ___ 29 g kept in vivarium conditions and fed a standard 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 s u p p l e m e n t e d 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 w e r e 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 d e t e r m i n e d at the beginning of the experiment, at 60 and 100 d, by m e a n s of a model 300 Perkin-Elmer atomic absorption spectrophotometer 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 proportion of 1:1 for Cu and 1:2 for Zn. Biolocjical Trace Element Research
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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
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Wad etal. Table 1 S e r u m Cu a n d Z n C o n c e n t r a t i o n (p,g/100 mL, M e a n Values + SD) in Control G r o u p (A), t h e G r o u p of A n i m a l s Fed a C h o l e s t e r o l - S u p p l e m e n t e d Diet (B), a n d the G r o u p Fed a C h o l e s t e r o l - S u p p l e m e n t e d Diet a n d Treated With C o p p e r Sulfate (C)
N
C u p,g %
Zn gg %
Zn/Cu
Initial
After 60 d
After 100 d
Initial
After 60 d
After 100 d
Initial
After 60 d
After 100 d
Group A (30)
121.2 +6.21
120.5 _+5.32
119.9 •
108.6 +6.82
108.9 +7.13
109.7 +4.25
0.90 --_0.07
0.91 +0.06
0.91 +0.04
Group B (30)
122.8 _+3.41
- 132.1 •
*'152.6 +4.8
111.5 _+2.04
102.3 _+4.12
*'82.1 +4.48
0.91 _+0.03
0.77 +0.05
**0.54 +0.03
Group C (30)
120.4 _+4,63
*'150.8 +4.05
*'152.8 _+4.33
109.1 _+8.24
105.9 -+3.12
103.3 +8.21
0.91 --0.07
*0.70 +0.05
~ 0.67 _+0.06
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, a n d Esterified S e r u m Cholesterol (mg/100mL, M e a n Values -+ SD) in Control G r o u p (A), the G r o u p Fed a C h o l e s t e r o l - S u p p l e m e n t e d Diet (B), a n d the G r o u p Fed a CholesterolS u p p l e m e n t e d Diet a n d Treated with C o p p e r Sulfate (C) Total s e r u m cholesterol mg/100mL
N
Esterified s e r u m cholesterol mg/100mL
Free s e r u m cholesterol mg/100mL
Initial
After 60 d
After 100 d
Initial
After 60 d
After 100 d
Initial
After 60 d
After 100 d
Group A (3O)
61.0 +2.9
66.0 _+4.2
68.2 •
36.3 _+3.3
41.8 +2.0
43.3 +3.7
24.7 -+2.3
24.2 _+3.5
25.0 +4.6
Group B (30)
60.2 _+7.2
76.9 +5.4
- ~'91.9 •
35.2 +3.4
53.4 -+4.2
**66.7 -+2.7
25.0 -+2.7
23.5 _+3.1
24.8 _+3.7
Group C (30)
61.3 -+6.1
71.7 _+7.9
76.6 •
36.0 _+2.1
48.8 +2.8
55.1 +3.9
25.3 23.4
22.3 _+3.6
21.5 +2.1
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 Biological Trace Element Research
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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).
JViorphologicalChanges C o m p a r e d to the normal aspect of the control group, (Fig. 1), after 60 d of high lipid diet, animals of group B s h o w e d 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 s u p p l e m e n t 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 atheromatous 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 h u m a n s dying with severe atherosclerotic lesions (13). Other authors Biological Trace Element Research
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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 diminished and minimal lipid sediments were revealed. f o u n d small Cu concentrations in the atherosclerotic tissue (14,15). Consequently there is no correlation b e t w e e n the blood- a n d tissue-Cu concentrations. It has been suggested that r e d u c e d Cu in tissues makes the cells more avid for cholesterol (16). Biological Trace Element Research
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Vlad et aL
In g r o u p C, administration of Cu sulfate reduces cholesterinemia. S e r u m Cu level a n d aortic Cu concentration also increase. In liver a n d k i d n e y s the Cu concentration increased only after 60 d of t r e a t m e n t with c o p p e r sulfate. After 100 d the values were nearly the same as in the control g r o u p . It is probable that d u r i n g this time the Cu shifted to the arterial tissues. As a consequence, the lipid infiltration in the arterial wall decreased. In conclusion, in experimental atherosclerosis the concentration of Cu in the a b d o m i n a l aorta a n d the liver decreases significantly. The a d m i n i s t r a t i o n of c o p p e r sulfate in these animals for 100 d i n d u c e d a significant increase of s e r u m Cu level, aortic a n d liver Cu concentration, a n d r e d u c e s s e r u m cholesterol concentration in c o m p a r i s o n with the g r o u p of animals fed a cholesterol-rich diet. In the aorta of animals treated with c o p p e r sulfate the e d e m a a n d lipid infiltration are less t h a n in the g r o u p fed only a h i g h lipid diet.
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