Copper and zinc concentrations in atherosclerotic plaque and serum in relation to lipid metabolism in patients with carotid atherosclerosis

Background/Aim. Some oligoelements are now investigated as possibly having a role in atherosclerosis. The aim of this study was to compare the concentrations of copper and zinc in the serum and carotid plaque and parameters of lipid metabolism in patients with different morphology of carotid atherosclerotic plaque. Methods. Carotid endarterectomy due to the significant atherosclerotic stenosis was performed in 91 patients (mean age 64 ± 7). The control group consisted of 27 patients (mean age 58 ± 9), without carotid atherosclerosis. Atheroscletoric plaques were divided into four morphological groups, according to ultrasonic and intraoperative characteristics. Copper and zinc concentrations in the plaque, carotid artery and serum were measured by atomic absorption spectrophotometry. Results. Serum copper concentrations were statistically significantly higher in the patients with hemorrhagic in comparison to those with calcified plaque (1.2 ± 0.9 μmol/L vs 0.7 ± 0.2 μmol/L, respectively; p = 0.021). Zinc concentrations were statistically significantly lower in plaques of the patients with fibrolipid in comparison to those with calcified plaques (22.1 ± 16.3 μg/g vs 38.4 ± 25.8 μg/g, respectively; p = 0.024). A negative significant correlation was found for zinc and triglycerides in the serum in all the patients (r = -0.52, p = 0.025). In the control group we also demonstrated a positive significant correlation for low-density lipoprotein cholesterol and copper in the serum (r = 0.54, p = 0.04). Conclusion. The data obtained in the current study are consistent with the hypothesis that high copper and lower zinc levels may contribute to atherosclerosis and its sequelae as factors in a multifactorial disease. Further studies are necessary in order to conclude whether high concentration of copper and zinc in the serum could be risk factors for atherosclesrosis.


Introduction
The positive correlation of elevated low-density lipoprotein (LDL) cholesterol serum levels and inverse correlation of high-density lipoprotein (HDL) cholesterol levels with the atherosclerotic process is a mainstay for research activities regarding pathophysiology and treatment of atherosclerotic clinical manifestation 1 .
In recent years investigators have focused their research activities on the role of several trace elements in the pathogenesis of atherosclerosis.Among trace elements, copper and zinc are well-documented as important modulating factors in cardiovascular homeostasis.Special research interest has been given to the relationship between copper and zinc serum and tissue concentrations and lipid status.Several studies pointed out a significant role of copper in LDL oxidation, however the exact mechanism is not yet understood 2 .
Copper has the pro-and anti-atherogenic (dual) effect 3 .Some experimental studies showed a positive correlation of copper deficiency with high total cholesterol levels and atherosclerotic changes on coronary arteries 4 .Low copper intake with copper defficiency and high dietary zinc/copper ratio leads to the increase in total serum cholesterol and development of atherosclerotic coronary disease 5 .Other studies implied that high copper serum levels could lead to the endothelial damage and the beginning of the atherosclerotic process 6 .
Zinc is an essential trace element for the normal membrane structure and function of important enzymes 7 .Studies reported that low zinc concentration may have an important role in the pathogenesis of atherosclerosis 8 .The effect of high zinc concentration on endothelial cells is not yet understood.Some evidence suggest that zinc can act as an endogenous protective factor against atherosclerosis by inhibiting the oxidation of LDL by cells or transition metals and protect the cell against cell-destabilizing agents such as inflammatory cytokines and polyunsaturated lipids 9 .
Through the interaction with endogenic basic fibroblast growth factor zinc potentiates endothelial and vascular smooth muscle cell proliferation which is an important part of intimal hyperplasia during the atherosclerotic process 10 .
Diversity and heterogeneity of atherosclerotic plaques implies that the atherosclerotic process is dynamic, complex and continuous.Numerous factors influence the morphology and growth pattern of different atherosclerotic plaques.
The aim of this study was to compare the concentrations of copper and zinc in the serum and carotid plaque and parameters of lipid metabolism in patients with different morphology of carotid atherosclerotic plaques.

Methods
The study group consisted of 118 subsequent patients (mean age 63 ± 8 years; 54.2% males) admitted to the Department of Vascular Surgery at Dedinje Cardiovascular Institute in Belgrade for carotid surgery.Carotid endarterectomy due to significant atherosclerotic stenosis and symptoms of cerebrovascular insufficiency was performed in 91 patients (mean age 64.2 ± 6.9 years, 62.6% males).The control group consisted of 27 patients (mean age 58.4 ± 9.3 years, 25.9% males) without carotid atherosclerosis which were operated due to the symptomatic kinking and coiling of carotid artery.
All the patients underwent neurological, cardiological and vascular surgeon exam, carotid artery ultrasound measurements and were treated surgically.Blood samples were drawn from all the patients before the surgery and sera were analyzed for the concentrations of lipid parameters (total cholesterol, HDL-and LDL-cholesterol and triglycerides), copper and zinc.
Tissue samples, atherosclerotic plaques from the atherosclerotic patients and normal carotid tissue from the control group patients were analyzed for the concentrations of copper and zinc.In the atherosclerotic subjects, atheroscletoric plaques were divided into four morphological groups, namely fibrolipid, hemorrhagic, ulcerated, and calcified plaque, according to ultrasonic and intraoperative characteristics 11 .
The study was approved by the Ethics Committee of the Dedinje Cardiovascular Institute.Written consent was obtained from all the patients before they entered the study.
Total cholesterol concentration in the serum was determined by the reaction with cholesterol-oxidase by using a diagnostic kit by Abbott.HDL-cholesterol in the serum was determined with the direct method by using a diagnostic kit by Abbott.Triglycerides in the serum were estimated by the reaction with glycerol-oxidase by using a diagnostic kit Abbott.LDL cholesterol was calculated according to the obtained levels of other lipid parameters noted above.
Copper and zinc concentrations in plaques, carotid artery tissue and the serum were estimated by means of flame atomic absorption spectrophotometry (AAS) using the Varian AA-5 instrument under the conditions recommended by the producer (acetylene/air flame, wavelenght: λCu = 324.75nm, λZn = 213.86nm).
Data are presented graphically as a box-plots, showing the mediana, minimum and maximum values.Differences in quantitative variables were assessed using the Mann-Whitney U test.The associations between the groups were analyzed by the Spearman's correlation.A p-value < 0.05 was considered statistically significant.Data were analyzed by SPSS version 10 (SPSS Inc, Chicago, Illinois, USA).

Results
Our patients cohort exhibited most of the classic atherosclerosis risk factors (Table 1).The percentage of hypertensive, smoking subjects was higher in the patients with carotid plaque than in the controls (p < 0.05).The presence of diabetes mellitus showed statistically significant differences between the patients and the controls (p < 0.05).
Atherosclerotic subjects were divided into four groups according to different morphology of analyzed atherosclerotic plaques 11 .The group I (22 patients; 18.6%) represented the patients with fibrolipid atherosclerotic plaques.The group II included the patients with hemorrhagic plaques (8 patients; 6.8%), the group III included the patients with ul- Values of the distribution of HDL-and LDL-cholesterol between the different plaques morphology groups and controls are presented in Figures 1 and 2, respectively.

Fig. 2 -Average low-density lipoprotein (LDL) cholesterol values in patients with different morphological groups of atherosclerotic plaques (for explanation see under Figure 1)
The distribution of copper concentration in plaque, carotid tissue and the serum are shown in Figures 3 and 4, respectively.Our study revealed a statistically significantly higher average serum copper concentration in the patients with hemorrhagic plaque (the group II) compared to the patients with calcified (the group IV) atherosclerotic plaques (1.2 μg/g ± 0.9 vs 0.7 μg/g ± 0.2, respectively; p < 0.05).The average values of copper concentrations in morphologically different atherosclerotic plaques and normal carotid tissue were similar.(for explanation see under Figure 1) No significant difference in the serum zinc concentrations was found among the groups (Figure 5).We found a statistically significantly lower average zinc plaque concen-   *p < 0.05 (statistically significant difference between the groups II and IV) (for explanation see under Figure 1) The negative and significant correlation coefficients were calculated for zinc and triglycerides in the selected patients (r = 0.52, p = 0.025) (data not showed).
In the control group we also demonstrated positive significant correlation coefficient for LDL-cholesterol and copper in the serum (r = 0.54, p = 0.04) (data not showed).

Discussion
Metal ions have been proposed as causative agents in a number of diseases, including atherosclerosis.The involvement of transition metals in atherosclerosis is controversial.Experimental studies have reported elevated levels of iron and copper in diseased human arteries but have often used methods that release metal ions from proteins 12 .Elevated levels of copper were also detected in carotid lesions 13 .Other studies do not support the hypothesis that elevated metal ion levels may be a major causative factor in aggravated atherosclerosis 14 .
Copper can promote low-density lipoprotein oxidation and the formation of macrophage-derived foam (lipid-laden) cells in vitro 15 .These elevated metal ion levels may therefore affect plaque stability and propensity to rupture.
Our study revealed a significantly higher copper concentration in the serum of patients with complicated hemorrhagic plaque in comparison to the morphological group IV (calcified plaque).There are no consistent data in the literature regarding the relationship between serum copper concentrations and the severity of atherosclerotic process 16 .
An Iranian study revealed a significantly higher serum copper concentration in patients with coronary heart disease of both sexes compared to controls 17 .
Diaz Romero et al. 18 determined equivalent serum copper values in patients with coronary heart disease and controls.Correspondingly to our results Alissa et al. 19 revealed no significant difference in both copper and zinc serum concentrations between the patients with established atherosclerosis and controls.
Our investigation showed a similar average copper concentration in atherosclerotic plaques and normal carotid tissue.The previous study of our group reported significantly higher copper concentrations in patients with ulcerated plaque in comparison to copper concentration in normal carotid tissue 20 .Serum copper concentrations are also associated with other coronary risk factors, including body mass index, levels of physical activity, serum HDL-cholesterol and C-reactive protein (CRP) 21 .
Polish authors 22 in their investigations demonstrated higher copper concentration in atherosclerotic plaques of patients with peripheral arterial disease in comparison to controls.
Our results show a trend of higher serum zinc concentration in the patients with fibrolipid and hemorrhagic plaques than in the patients in other morphology groups of atherosclerotic plaques.We found no significant difference between serum zinc levels in atherosclerotic and normal subjects.We found a significantly lower average zinc plaque concentration in the patients with fibrolipid plaques in comparison to the patients with calcified plaques.Stadler et al. 23 found elevated levels of zinc in human atherosclerotic advanced lesions carotid and abdominal artery compared to healthy tissue or early lesions.The control subjects had no significantly higher plaque zinc levels compared to noncalcified atherosclerotic plaque patients which is in accordance with our earlier investigation 20 .
Recent studies reported lower zinc serum levels in patients with different clinical form of atherosclerosis compared to controls and suggested that protective effect of zinc accumulation in atherosclerotic plaque could be associated with lesion calcification 23,24 .Patients with the established coronary artery disease had significantly higher serum CRP and lower serum zinc compared to both patients without coronary artery disease and healthy controls 16 .Zinc deficiency has been associated with the development of atherosclerosis 23 .
The presented higher proportion of male subjects in atherosclerotic groups and female subject in patients with kinking and coiling carotid artery is in accordance with the results of earlier studies 25 .The atherosclerotic subjects in our study had higher LDL cholesterol and lower HDL cholesterol serum levels in comparison to the control subjects.Previous studies also demonstrated a significant positive correlation of LDL values and negative correlation of HDL values with the severity of the carotid atherosclerotic process 26 .The investigators from the "Tromso study" reported a strong positive correlation of HDL values and reduction of atherosclerotic plaque progression, higher plaque echogenity and in some cases a reduction in plaque size 27 .
We found a significant negative correlation between serum zinc concentration and triglycerides.Serum copper concentrations were positively associated with fasting serum triglycerides in Iranian patients 16 .In the control group we demonstrated a positive significant of LDL cholesterol and copper concentration in the serum.
Human studies investigating the effect of copper and zinc status on cholesterol metabolism are very limited 28 .In an adult African-American community females taking either zinc supplements had higher HDL-cholesterol values than nonsupplementing females 29 .
Serum zinc and copper levels were both significantly lower in individuals with normal versus high levels of LDLcholesterol in a large Iranian population sample 30 .One of the theory implies that hypercholesterolemia and endothelial damage lead to peroxynitrite formation, decrease in pH and increase in copper ion release from ceruloplasmin.This process enhance LDL oxidation and plaque growth.Several studies failed to confirm a significant correlation between oligoelements and lipoprotein fractions 28 .Iranian authors 17 reported no significant relationship between copper and zinc serum concentration and different lipoprotein fractions in atherosclerotic patients.

Conclusion
Overall, the data obtained in the current study are consistent with the hypothesis that copper and zinc may contribute to atherosclerosis and its sequelae as factors in a multifactorial disease.Registered differences in oligoelements between different morphological groups should be further evaluated in clinical settings of different oligoelements intake.

Fig. 1 -
Fig. 1 -Average high-density lipoprotein (HDL) cholesterol values in patients with different morphological groups of atherosclerotic plaques.Groups of patients: I -with fibrolipid atherosclerotic plaque; II -with hemorrhagic plaque; III -with ulcerated atherosclerotic plaque; IV -with calcified atherosclerotic plaque.

Fig. 3 -
Fig.3-Average plaque copper concentration in the different groups of patients (for explanation see under Figure1).

Fig. 4 -
Fig. 4 -Average serum copper concentration in the different groups of patients.*p < 0.05 (statistically significant difference between the groups II and IV).(for explanation see under Figure 1) tration in the patients with fibrolipid plaque (the group I) in comparison to the patients with calcified (the group IV) plaques (22.1 ± 16.3 μg/g vs 38.4 ± 25.8 μg/g respectively; p = 0.024), (Figure6).

Table 1 Baseline characteristics of the patients with carotid atherosclerosis (the study group) and the control group of patients
Results are given as percentage of patientsTasić MN, et al.Vojnosanit Pregl 2015; 72(9): 801-806.