European Journal of Pharmacology 738 (2014) 326–331
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Cardiovascular pharmacology
The influence of selected antihypertensive drugs on zinc, copper, and iron status in spontaneously hypertensive rats Joanna Suliburska a,n, Paweł Bogdanski b, Hieronim Jakubowski c,d,e a
Department of Human Nutrition and Hygiene, Poznan University of Life Sciences, Poznan, Poland Department of Internal Medicine, Metabolic Disorders and Hypertension, University of Medical Sciences, Poznan, Poland c Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, Poznan, Poland d Institute of Bioorganic Chemistry, Poznan, Poland e Department of Microbiology & Molecular Genetics, Rutgers University-New Jersey Medical School, International Center for Public Health, Newark, NJ, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 February 2014 Received in revised form 25 May 2014 Accepted 2 June 2014 Available online 11 June 2014
Mineral homeostasis in hypertensive patients may be affected by hypotensive drugs. The aim of this study was to assess the influence of selected antihypertensive drugs on mineral homeostasis in a rat model of hypertension. Eight-week-old male spontaneously hypertensive rats (SHRs) were treated with perindopril, metoprolol, indapamide, amlodipine, or no drug for 45 days. In another experiment, the SHRs were treated with indapamide or amlodipine in the presence of zinc and copper gluconate supplement. Lipids, glucose, and insulin levels along with superoxide dismutase and catalase activities were assayed in serum. Iron, zinc, and copper concentrations in serum, erythrocytes, and tissues were determined using the flame atomic absorption spectrometry. Blood pressure was measured using a tailcuff plethysmograph. Treatment with indapamide and amlodipine was found to significantly lower zinc levels in serum, erythrocytes, livers, and spleens of the SHRs, as well as copper levels in the kidneys, compared with the control no-drug group. A markedly higher concentration of glucose was found in the indapamide-treated rats. Supplementing the indapamide-treated SHRs with zinc and copper gluconate resulted in a significant decrease in both systolic and diastolic blood pressure, and also lowered serum glucose and triglyceride concentrations and HOMA (homeostasis model assessment-insulin resistance) values. The results show that indapamide and amlodipine disturb zinc and copper homeostasis in SHRs. Supplementation with zinc and copper restores mineral homeostasis in SHRs treated with indapamide and amlodipine, and also corrects metabolic imbalances while improving the antihypertensive efficiency of indapamide. & 2014 Elsevier B.V. All rights reserved.
Keywords: Copper Hypotensive drugs Iron Spontaneously hypertensive rats Zinc Chemical compounds studied in this article: Perindopril (PubChem CID: 107807) Amlodipine (PubChem CID: 2162) Indapamide (PubChem CID: 3702) Metoprolol (PubChem CID: 4171) Zinc gluconate (PubChem CID: 158040) Copper gluconate (PubChem CID: 10692)
1. Introduction Hypertension is a common disease in Western societies, and is usually treated with hypotensive drugs that are prescribed longterm. Antihypertensive drugs include angiotensin-converting enzyme inhibitors (ACEi), diuretics, calcium-channel blockers, beta blockers, and angiotensin receptor blockers (ARB); alpha blockers are also frequently used. These drugs are taken alone (monotherapy) or in combination (polytherapy). The long-term use of hypotensive drugs may cause side effects, such as inverse disturbances in electrolyte homeostasis, which is relatively common with the use of diuretics and ACEi (Braun and Rosenfeldt, 2003). n Correspondence to: Department of Human Nutrition and Hygiene, Poznan University of Life Sciences, ul. Wojska Polskiego 31, 60-624 Poznan, Poland. Tel.: þ 48 61 8487334; fax: þ48 61 8487332. E-mail address: [email protected] (J. Suliburska).
http://dx.doi.org/10.1016/j.ejphar.2014.06.003 0014-2999/& 2014 Elsevier B.V. All rights reserved.
In clinical and experimental studies, including our own, an association is observed between disordered sodium, potassium, magnesium, and calcium homeostasis and essential hypertension (Joosten et al., 2013; Pikilidou et al., 2007). It has been suggested that long-term antihypertensive therapy affects zinc pharmacokinetics, particularly in patients with conditions associated with disturbances of zinc homeostasis (Braun and Rosenfeldt, 2003). Antihypertensive therapy may significantly affect mineral status, and may require systematic monitoring (Pikilidou et al., 2007; Suliburska et al., 2011). Moreover, disorders in mineral status may impact lipid and glucose metabolism, and also mineral-dependent enzyme activity, such as that of superoxide dismutase (SOD) and catalase (CAT), in the body (Suliburska et al., 2011; Zuo et al., 2006). Drugs generally are capable of influencing the metabolism of trace elements in many ways from intestinal absorption to bioavailability and elimination. It is not known whether mineral supplementation or dietary modification might be an optimal
J. Suliburska et al. / European Journal of Pharmacology 738 (2014) 326–331
treatment for hypertensive patients. However, in studies of mineral homeostasis in patients, the drugs used are rarely taken into consideration. Because the interactions between hypotensive drugs and nutrients are poorly understood, we have examined the effect of perindopril, metoprolol, indapamide, and amlodipine on iron, zinc, and copper homeostasis in the SHR model. We also studied the effect of the combined mineral supplements and drugs on mineral homeostasis in SHRs.
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Table 2 The amount of supplements in the diet in the experiment with supplementation. Group
Supplement (g/kg diet)
Total zinc Total copper (mg/kg) (mg/kg)
Zinc gluconiate Copper gluconiate C2 (n¼10) ID2 (n ¼10) AM2 (n ¼10)
– 0.627 0.627
– 0.129 0.129
70 160 160
13 31 31
C-control group, ID2-group with indapamide and zinc and copper supplements. AM2-group with amlodipine and zinc and copper supplements.
2. Materials and methods 2.1. Animals Eight-week-old male spontaneously hypertensive rats (SHRs) (Charles River Laboratories, Germany), derived from Wistar Kyoto rats with elevated blood pressure at the Kyoto School of Medicine, were used. The mean weight of the rats was 195 721 g. The animals were housed individually in stainless steel cages coated with metal-free enamel and kept under cycles of 12 h light and 12 h dark. The room temperature was maintained at 21 71 1C with 55–65% humidity. The animal procedures were approved by the local bioethics committee (approval no. 49/2009).
The second experiment (with supplementation) was designed on the basis of the first experiment. In this experiment drugs were used with potential influence on mineral status. Thirty animals were randomly assigned to three groups of 10 animals each: control diet (C2); diet with indapamide, zinc, and copper gluconate (ID2); and diet with amlodipine, zinc, and copper gluconate (AM2). The supplemented diets were prepared by mixing an appropriate proportion of the diet with the supplements (USP Merck). The proportions of the diet and supplements in each mix are shown in Table 2. The procedure of the second experiment followed that of the first. The experiments were begun following a 5-day period to allow the rats to adapt to laboratory conditions.
2.2. Experimental design
2.3. Blood-pressure measurements
Two experiments were performed. In the first (without supplementation), SHRs were randomly assigned to five groups of 10 animals each: the control, diet without drugs (C1), diet with perindopril (PR1), diet with metoprolol (MT1), diet with indapamide (ID1), and diet with amlodipine (AM1). The rats were fed a standard diet (maintenance diet for rats 1320, Altromin). The full composition of the diet is presented in Table 1. All rats were provided ad libitum diet and distilled water for 45 days. Dietary intake was recorded daily. Body weight was recorded each week prior to food distribution. In the diet of the noncontrol groups, perindopril, metoprolol, indapamide, and amlodipine were added at a rate of 0.2, 3.0, 0.03, and 0.2 mg/kg body mass of rat, respectively. The drug was administered in the diet, combined with chow, to which the rats had free access. Fresh solutions were prepared every day. The drug concentrations were adjusted so that the doses (calculated as milligrams per kilogram per day) were kept constant, regardless of diet intake and body weight. In this study, relatively low doses of these drugs were used.
Blood pressure was measured with a tail-cuff plethysmograph using a blood-pressure measuring system (MODEL MK-1030, Muromachi Kikai). The systolic and diastolic blood pressure was measured following 15 min warming at 37 1C in an animal holder made of dark brown acryl, allowing blood-pressure measurement under relatively stress-free conditions. An average of five readings was recorded for each animal. The rats were first habituated to the measurement device and remained unperturbed in the chamber throughout the inflation–deflation cycles.
Table 1 The composition of the base diet. Ingredient
Amount Ingredient
Total energy (kcal/kg) 2844 Total protein (% of energy) 24 Total fat (% of energy) 11 Total carbohydrate (% of energy) 65 Protein (g/100 g) 19 Fat (g/100 g) 4 Fiber (g/100 g) 6 Vitamin A (IU) 1500 Vitamin D3 (IU) 600 Vitamin B1 (mg/kg) 18 Vitamin B2 (mg/kg) 12 Vitamin B6 (mg/kg) 9 vitamin B12 (μg/kg) 24 vitamin C (mg/kg) 36 vitamin K3 (mg/kg) 3 Vitamin E (mg/kg) 75 Folic acid (mg/kg) 2
Amount
Biotin (μg/kg) 60 Nicotinic acid (mg/kg) 36 Pantothenic acid (mg/kg) 21 Choline chloride (mg/kg) 600 Calcium (g/kg) 9 Phosphor (g/kg) 7 Magnesium (g/kg) 3 Sodium (g/kg) 2 Potassium (g/kg) 1 Iron (mg/kg) 165 Manganium (mg/kg) 75 Zinc (mg/kg) 70 Copper (mg/kg) 13 Iodium (mg/kg) 1.5 Selenium (mg/kg) 0.6 Cobalt (mg/kg) 0.3
2.4. Tissue and serum collection The animals were fasted for 12 h prior to termination of the experiment. At the end of the experimental period, the animals were weighed and killed. The rats were anesthetized with a sodium thiopental injection (40 mg/kg body weight) and killed by cardiac puncture. The tissues (liver, spleen, kidneys, heart, pancreas, gonads) were dissected, weighed, and stored frozen (at 70 1C) for mineral content analysis. Each blood sample obtained by cardiac puncture was divided between a serum separator tube and a tube containing heparin sodium. The samples in the serum separator tubes were left undisturbed at room temperature for 30 min, and then centrifuged for 15 min at 1048 g at 4 1C; the supernatant was removed and stored at 70 1C. To isolate erythrocytes, whole blood in heparin sodium-containing tubes was centrifuged for 15 min at 1048 g at 4 1C, and the plasma was removed. Blood cells were washed thrice with 5 ml of 0.9% saline solution and centrifuged at 2493 g for 10 min at 4 1C. Following each centrifuging, saline solution was separated and the erythrocyte mass was placed in demineralized Eppendorf tubes and stored at 70 1C for minerals analysis. 2.5. Biochemical measurements Total cholesterol (TC), triglyceride (TG), and fasting glucose levels in serum were measured using commercial kits (Randox Laboratory Ltd, UK) at a diagnostic laboratory in Poznań, Poland. The concentration of cholesterol and triglycerides in serum was
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assayed using the enzymatic method. The concentration of glucose in the blood serum was estimated using the glucose oxidase method (Clinical and Laboratory Standards Institute/NCCLS, 2004). Serum insulin was determined with the radioimmunoassay method, using a rat insulin RIA kit (Insulin RIA Kit, Linco Research, USA). Insulin resistance was evaluated according to the homeostasis model assessment-insulin resistance (HOMA-IR) protocol: HOMA index ¼[fasting insulin (Um/l) fasting glucose (mmol/l)]/ 22.5. Hemoglobin was determined using the cyanmethemoglobin method (Mahoney et al., 1993). Serum superoxide dismutase (SOD) and catalase (CAT) activities were measured using an immunoenzymatic SOD and CAT activity assay kit (Cayman Chemicals, USA). 2.6. Determination of minerals The iron, zinc, and copper contents of the serum, erythrocytes, and tissues were determined following digestion in 65% (w/w) spectra pure HNO3 (Merck) in the Microwave Digestion System (MARS 5, CEM Corp., USA). Thereafter, the concentrations of iron, zinc, and copper in the mineral solutions were measured using the flame atomic absorption spectrometry method (AAS-3, Carl Zeiss, Jena, Germany). The mineral contents of serum and tissues were determined at the following wavelengths: for iron, 248.3 nm; for zinc, 213.9 nm; for copper, 324.8 nm. The accuracy of the method was verified with certified reference materials (bovine liver-trace elements, NIST-1577C, CERT and seronorm TRACE ELEMENTS Whole Blood L-2), and was 95–98% for iron, 95–96% for zinc, and 99–103% for copper. 2.7. Statistical analysis Detailed statistical analysis was performed using Statistica for Windows 10.0 (StatSoft, Poland). The results were expressed as arithmetic means with standard errors. One-way analysis of variance (ANOVA), followed by a post-hoc Turkey's test, were used to compare the data between groups. Student's t-test was used to compare the two means between groups from the first and the second experiment. The significance was set at the P o0.05 level.
3. Results The results of the first experiment, which did not involve supplementation, are shown in Tables 3–5. It was found that hypotensive treatment led to a decrease in both systolic (SBP) and diastolic blood pressure (DBP), but a
significant decrease was observed only in the groups given perindopril (SBP: 23.3%; DBP: 27.9%) and metoprolol (SBP: 19.7%; DBP: 30.9%), compared with the control no-drug group. Markedly higher concentrations of glucose in the ID1 group (þ11.5%), and markedly lower concentrations of triglycerides in the MT1 group ( 19.4%), were observed. Treatment with the diuretic indapamide or with the calcium-channel blocker amlodipine resulted in significant decreases in zinc in serum (ID1: 14.3%; AM1: 15.1%), erythrocytes (ID1: 7.0%; AM1: 5.0%), livers (ID1: 12.8%; AM1: 18.0%) and spleens (ID1: 9.0%; AM1: 10.2%) of the SHRs. Markedly lower liver copper ( 40.4%) and higher kidney iron ( þ15.0) in ID1 rats were observed. Perindopril markedly increased the concentration of iron in the rats' livers (þ 11.4%). Significantly lower concentrations of copper in the kidneys were found in the MT1 ( 13.3%), ID1 ( 17.8%), and AM1 groups ( 17.8%). The results of the second experiment, which employed supplementation, are shown in Tables 6–8. When the indapamidetreated (ID2) and amlodipine-treated (AM2) SHRs were supplemented with zinc and copper gluconate, significant decreases were observed in systolic (ID2: 19.4%; AM2: 20.0%) and diastolic blood pressure (ID2: 29.3%; AM2: 35.1%), while decreases were seen in serum glucose (ID2: 17.2%; AM2: 7.8%) and triglyceride (ID2: 30.0%; AM2: 25.0%) concentrations in the blood. The HOMA value for the ID2 group was significantly lower (ID2: 46.7%) than that of the C2 group. No significant differences were observed in tissue mineral concentrations between the study groups (ID2 and AM2) and the control group (C2). The results of the second experiment (with supplementation) were compared with the results of the first experiment (without supplementation). It was shown that indapamide combined with zinc and copper supplementation significantly decreased systolic and diastolic blood pressure (SBP: 14.5%, DBP: 15.9%), serum glucose ( 22.1%), triglycerides ( 22.2%) and HOMA ( 36.0%), and increased serum zinc level (17.6%) and zinc and copper concentration in erythrocytes (15.8% and 37.3% respectively), compared with ID1 group. In the group treated with indapamide and with supplementation, markedly higher concentrations of zinc and copper in the livers (9.3% and 31.1%), spleens (25.9%, 71.4%), and kidneys (39.9%, 32.4%) were found, as were markedly higher levels of zinc in the hearts (10.6%). In turn, the concentration of iron in livers, kidneys, hearts, and pancreases was significantly lower ( 19.7%, 21.4%, 25.8%, 14.9) in group ID2 than ID1. In the rats treated with amlodipine and with supplementation, significantly higher zinc concentrations in serum (18.8%) and erythrocytes (28.2%) were seen, compared to the rats without supplementation. Markedly higher concentrations of zinc and
Table 3 Blood pressure and biochemical parameters in rats in the experiment without supplementation. Parameter
SBP (mmHg) DBP (mmHg) Glucose (mmol/l) Insulin (pmol/l) HOMA Cholesterol (mmol/l) Triglyceride (mmol/l) SOD (U/ml) CAT (nmol/min/ml)
Groups C1 (n¼10)
PR1 (n¼10)
MT1 (n¼ 10)
ID1 (n ¼10)
AM1 (n ¼10)
197.2 7 27.7 123.9 7 15.7 6.17 0.6 132.8 7 41.4 5.03 7 1.53 1.127 0.20 0.36 7 0.01 6.92 7 0.85 44147 1040
151.3 716.4a 89.3 725.7a 6.5 70.9 139.5 753.3 5.63 72.42 1.17 70.19 0.39 70.03 6.15 71.20 3951 7808
158.3 7 31.3a 85.6 7 29.4a 5.9 7 1.0 124.07 39.6 4.34 7 1.58 1.017 0.13 0.29 7 0.05a 6.55 7 0.80 36067 912
180.2 720.7 101.1 719.8 6.8 70.6a 107.0 720.7 4.46 70.89 1.05 70.14 0.36 70.07 6.72 71.06 3371 7950
170.27 30.4 103.6 7 26.9 6.17 0.6 136.3 7 50.3 5.147 1.80 1.047 0.13 0.34 7 0.07 6.83 7 0.89 4898 7 1055
C1-control group, PR1-group with perindopril, MT1-group with metoprolol, ID1-group with indapamide, AM1-group with amlodipine, SBP-systolic blood pressure, DBP-diastolic blood pressure, HOMA-insulin resistance index, SOD-superoxide dismutase, CAT-catalase. a
P o0.05 compared to C1.
J. Suliburska et al. / European Journal of Pharmacology 738 (2014) 326–331
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Table 4 Minerals concentration in serum and erythrocytes in the experiment without supplementation. Parameter
Groups C1 (n¼ 10)
PR1 (n¼ 10)
MT1 (n¼10)
ID1 (n ¼10)
AM1 (n ¼10)
Serum (μmol/l) Iron Zinc Copper
23.3 7 1.1 11.9 7 0.6 13.3 7 0.6
23.4 7 1.0 12.0 7 0.6 13.2 7 0.6
23.9 71.1 12.1 70.6 13.0 70.6
24.17 1.2 10.2 7 0.5a 12.6 7 0.7
23.9 7 1.2 10.17 0.6a 12.8 7 0.6
Erythrocytes Iron (μmol/g Hb) Zinc (μmol/g Hb) Copper (nmol/g Hb)
41.9 7 2.0 0.417 0.03 40.0 7 3.3
44.6 7 2.2 0.40 7 0.02 41.2 7 5.2
46.1 72.3 0.43 70.09 44.1 74.1
45.7 7 2.3 0.38 7 0.03a 36.2 7 2.2b
45.17 2.7 0.39 7 0.06a 39.3 7 3.8
C1-control group, PR1-group with perindopril, MT1-group with metoprolol, ID1-group with indapamide, AM1-group with amlodipine. a b
P o0.05 compared to C1. Po 0.05 compared to MT1.
Table 5 Iron, zinc and copper concentration in tissues in the experiment without supplementation (μg/g wet wt.). Parameter
Groups C1 (n¼10)
PR 1(n ¼10)
MT 1(n¼ 10)
ID1 (n¼ 10)
AM 1(n¼ 10)
Liver Iron Zinc Copper
158.6 7 9.1 40.5 7 1.5 5.2 7 0.2
176.7 7 10.3a 41.0 7 2.4 4.8 7 0.3
164.2 75.4 40.1 72.2 4.8 70.3
165.2 7 6.8 35.3 7 1.5a 3.17 0.4a
167.7 7 13.6 33.27 1.7a 5.17 0.4
Spleen Iron Zinc Copper
727.0 7 96.8 25.5 7 2.6 1.8 7 0.6
737.9 7 105.8 24.27 1.3 1.4 7 0.6
661.2 7103.1 24.371.0 1.7 70.4
664.6 7 105.6 23.2 7 1.0a 1.4 7 0.5
653.4 7 97.7 22.9 7 0.9a 1.4 7 0.4
Kidney Iron Zinc Copper
61.4 7 6.9 20.0 7 1.1 4.5 7 0.1
68.57 8.0 20.4 7 0.5 4.17 0.2
68.8 73.8 20.6 71.1 3.9 70.2a
70.6 7 5.4a 20.3 7 0.8 3.7 7 0.3a
65.2 7 5.3 20.6 7 1.2 3.7 7 0.3a
Heart Iron Zinc Copper
83.17 7.0 26.8 7 1.5 5.7 7 0.2
91.7 7 14.2 26.5 7 2.6 6.0 7 0.7
85.7 712.3 26.2 71.7 5.8 70.6
90.6 7 6.9 25.4 7 1.6 5.5 7 0.3
81.0 7 10.1 25.4 7 1.7 5.4 7 0.5
Pancreas Iron Zinc Copper
25.17 2.8 26.9 7 1.5 1.17 0.2
27.0 7 3.3 26.7 7 2.3 1.2 7 0.3
27.2 73.5 28.6 74.1 1.2 70.2
28.17 2.4 29.8 7 3.9 1.17 0.2
26.7 7 1.8 28.4 7 4.1 1.3 7 0.2
Gonads Iron Zinc Copper
22.9 7 1.4 30.8 7 2.2 1.9 7 0.2
24.47 1.0 32.4 7 1.2 1.8 7 0.2
22.5 71.6 31.1 72.7 1.8 70.1
23.5 7 1.8 29.5 7 0.9 1.7 7 0.2
23.3 7 2.2 30.0 7 1.0 1.6 7 0.2
C1-control group, PR1-group with perindopril, MT1-group with metoprolol, ID1-group with indapamide, AM1-group with amlodipine. a
P o0.05 compared to C1.
copper were also observed in the kidneys (31.1%, 24.3%) and hearts (15.4%, 9.4%) of in group AM2, compared with those of AM1. Moreover, zinc concentration in the livers (20.5%) and spleens (25.8%) was markedly higher in the AM2 group than in AM1. Treatment with amlodipine and supplementation decreased iron levels in the livers ( 18.7%), kidneys ( 13.8%), and pancreases ( 13.9%).
4. Discussion The influence of indapamide and amlodipine on zinc and copper homeostasis in SHRs is a new finding of this study. Furthermore, to the best of our knowledge, this study is the first experimental demonstration of a positive outcome on metabolic and mineral homeostasis in SHRs associated with zinc and copper supplementation during indapamide therapy.
Our results suggest that hypotensive drugs disturb zinc and, to a lesser extent, copper homeostasis in SHRs. Decreases in zinc levels in serum, erythrocytes, the liver, and the spleen during the ID and AM treatments were observed in the absence of any significant decrease in blood pressure in the ID-treaded and AMtreated animals. The decrease in zinc was accompanied by an increase in glucose serum levels in the ID-treated SHRs. Moreover, our results suggest that a combination of indapamide or amlodipine therapy with zinc and copper supplementation restores mineral homeostasis. In the case of indapamide, there is also a positive effect on lipid metabolism, carbohydrate metabolism, and blood pressure. The results obtained in the present study suggest that the interaction between zinc and hypotensive drugs exerts a pharmacokinetic effect, and may be caused by the inhibition of renal zinc reabsorption, resulting in zincuria (Braun and Rosenfeldt, 2003; Samaras et al., 2013). It is possible that antihypertensive drugs
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Table 6 Blood pressure and biochemical parameters in rats in the experiment with supplementation. Parameter
Groups C2 (n¼ 10)
SBP (mmHg) DBP (mmHg) Glucose (mmol/l) Insulin (pmol/l) HOMA Cholesterol (mmol/l) Triglyceride (mmol/l) SOD (U/ml) CAT (nmol/min/ml)
191.1 719.0 120.3 713.8 6.4 70.2 129.1 751.6 5.29 72.30 1.25 70.09 0.40 70.05 6.85 70.81 4448 71050
Parameter ID2 (n ¼10)
AM 2(n ¼10) a,A
154.0 7 25.2 85.0 7 16.7a,A 5.3 7 0.1a,A 86.1 7 49.9 2.85 7 1.56a,A 1.357 0.28 0.28 7 0.01a,A 6.94 7 0.80 3860 7 866
a
152.9 7 24.7 78.17 25.7a 5.9 7 0.4a 120.357 48.2 4.36 7 1.43 1.36 7 0.14 0.30 7 0.06a 6.98 7 0.86 43507 982
C2-control group, ID2-group with indapamide with Zn and Cu supplements. AM2-group with amlodipine with Zn and Cu supplements, SBP-systolic blood pressure. DBP-diastolic blood pressure, HOMA-insulin resistance index, SOD-superoxide dismutase, CAT-catalase. a
P o0.05 compared to C2. A Po 0.05 compared to ID1.
Table 7 Minerals concentration in serum and erythrocytes in the experiment with supplementation. Parameter
Groups C2 (n ¼10)
ID2(n¼ 10)
AM2 (n¼10)
Serum (μmol/l) Iron Zinc Copper
23.5 7 1.1 11.8 7 0.61 13.2 7 0.6
23.2 7 1.1 12.0 7 0.6A 13.0 7 0.6
23.9 71.2 12.0 70.6B 13.2 70.6
Erythrocytes Iron (μmol/g Hb) Zinc (μmol/g Hb) Copper (nmol/g Hb)
45.7 7 5.7 0.46 7 0.11 40.6 7 3.9
47.1 7 4.2 0.44 7 0.11A 49.7 7 4.9A
45.8 72.6 0.50 70.16B 33.9 74.8a
C2-control group, ID2-group with indapamide with Zn and Cu supplements. AM2-group with amlodipine with Zn and Cu supplements. a A B
Table 8 Minerals concentration in tissues in the experiment with supplementation (μg/g wet wt.).
P o0.05 compared to ID2. Po 0.05 compared to ID1. Po 0.05 compared to AM1.
affect zinc reabsorption in the kidney by decreasing renal Na/H antiporter activity. Interestingly, angiotensin II is a potent inducer of Na/H antiporter, and the synthesis of this hormone depends on the activity of angiotensin-converting enzyme (ACE) (Reeves and O’Dell, 1986). It is known that thiazides inhibit NaCl transport in the distal tubule, and may affect zinc reabsorption through this mechanism (Braun and Rosenfeldt, 2003; Samaras et al., 2013). Indapamide is a thiazide-like diuretic. In some human studies, it has been found that thiazide diuretics may increase zinc urinary excretion; in some patients, decreased serum zinc levels in serum have also been observed (Braun and Rosenfeldt, 2003). The results of clinical studies have revealed zinc disturbances caused by commonly prescribed diuretics. Increases in urinary zinc and decreases in both plasma and erythrocyte zinc levels were seen. Although, in the present study, urinary zinc was not assayed, the decreased levels of zinc in the analyzed rat tissues were most likely caused by the loss via urinary excretion. This is supported by the fact that no accumulation of zinc was observed in any of the analyzed tissue following treatment with indapamide and amlodipine. Our results show the relation between zinc homeostasis and blood pressure. It is suggested that disturbances in zinc homeostasis may affect blood pressure. Tubek (2007) indicated that low zinc concentrations in the extracellular space lead to less efficient
Groups C2 (n¼ 10)
ID2 (n¼ 10)
AM2 (n¼ 10)
Liver Iron Zinc Copper
135.879.3 38.7 70.7 4.2 70.2
132.7 7 11.2A 38.6 7 2.4A 4.5 7 0.7A
136.3 7 18.1B 40.0 7 2.1B 4.5 7 0.4
Spleen Iron Zinc Copper
638.3 777.8 28.4 71.2 2.2 70.4
631.3 7 94.7 29.2 7 2.0A 2.4 7 0.8A
635.97 43.7 28.8 7 0.8B 1.9 7 0.4
Kidney Iron Zinc Copper
56.8 76.2 28.3 71.2 4.7 70.3
55.5 7 2.4A 28.4 7 1.7A 4.9 7 0.5A
56.2 7 7.5B 27.0 7 1.2B 4.6 7 0.2B
Heart Iron Zinc Copper
69.2 75.7 27.7 72.1 5.8 70.3
67.2 7 6.9A 28.17 1.4A 5.8 7 0.6
70.27 9.0 29.3 7 2.5B 5.9 7 0.3B
Pancreas Iron Zinc Copper
23.4 74.3 26.4 73.4 1.3 70.3
23.9 7 0.7A 25.9 7 2.0 1.3 7 0.2
23.0 7 1.5B 26.6 7 2.5 1.2 7 0.3
Gonads Iron Zinc Copper
20.1 71.6 28.0 70.6 1.6 70.1
20.2 7 0.9A 28.5 7 1.0 1.7 7 0.2
20.7 7 2.6 29.0 7 1.2 1.7 7 0.3
C2-control group, ID2-group with indapamide with Zn and Cu supplements. AM2-group with amlodipine with Zn and Cu supplements. A B
Po 0.05 compared to ID1. P o0.05 compared to AM1.
blocking of calcium channels, favoring the movement of calcium into the cells and its accumulation there, leading to increased blood pressure. In our study, the lower zinc tissue concentration in the groups treated with indapamide and amlodipine was associated with slightly decreased blood pressure, while in the groups with perindopril and metoprolol (which lacked disturbed zinc homeostasis), the blood pressure was reduced. Moreover, the restoration of zinc content through diet supplementation caused a decrease in blood pressure in rats that was not observed in the nonsupplemented animals. The disorder in copper homeostasis that was observed in the SHRs following treatment with indapamide and amlodipine may occur because of metabolic interrelationships between zinc, copper, and iron. The decrease in the zinc level probably affected iron homeostasis (manifested as a slight increase in the liver and kidney iron levels), and the change in the concentration of these minerals may be related to the change in the tissue copper level (Arredondo and Nunez, 2005). The concentration of copper in serum and erythrocytes was not changed by hypotensive drugs, which is consistent with the results of clinical trials (da Cunha et al., 2002). In this study, metoprolol also influenced copper levels in the kidneys of rats. In some studies, it has been found that antihypertensive drugs disturb homeostasis of electrolytes and other minerals through alterations in kidney or intestine mineral reabsorption, as well as through their chelating properties and by causing changes in the reabsorption of minerals from the system blood to the tissues (Braun and Rosenfeldt, 2003; Samaras et al., 2013). The mechanism involved in the change in kidney copper concentration as a result of metoprolol treatment may be similar. The decrease in copper concentration in the kidney was the only change in the mineral status of rats treated with metoprolol, and
J. Suliburska et al. / European Journal of Pharmacology 738 (2014) 326–331
this change was associated with a decrease in the triglyceride level in serum. These results confirm the positive correlation between copper and triglycerides observed in other studies (Suliburska et al., 2011). It should be noted that disorders in zinc and copper homeostasis caused by hypotensive drugs may produce a wide range of negative effects in the body. It is known that zinc deficiency may predispose to glucose intolerance and insulin resistance, while copper deficiency leads to hypercholesterolemia and impaired endothelium-dependent arterial relaxation (Ghayour-Mobarhan et al., 2009). In the present study, we found that the lower status of zinc in SHRs treated with indapamide was associated with higher serum glucose concentrations. This could be caused by a decrease in the tolerance of glucose, due to the lower level of zinc in the rats, or to a lower level of insulin in serum, or both. It is known that zinc plays a major role in the synthesis and action of insulin. Zinc is a component of many enzymes, and is involved in the synthesis, storage, and release of insulin (Hashemipour et al., 2009). In the ID1 group, a slightly lower level of insulin was observed than in the control group. The supplementation of the diet with zinc and copper in SHRs treated with indapamide resulted in a significant reduction in triglycerides, glucose, and blood pressure. The combination of this drug with zinc and copper supplements increased the effectiveness of the treatment, and reduced the side effects of the drug. The significant decrease in the blood pressure of the rats treated with both indapamide and zinc and copper supplementation may be caused by the increase in SOD activity (although this increase did not reach statistical significance). In this study, we also investigated perindopril and observed a significantly higher iron concentration in the liver of rats on this drug. Similar to captopril (the first ACE inhibitor approved for use in patients), perindopril reduces blood pressure without adverse effects on serum glucose, insulin, or lipids in SHRs (Swislocki et al., 1999). Several studies have shown that, during treatment, perindopril had a persistent dose-dependent effect on blood pressure (Thybo et al., 1994). Skrzypczak et al. (2010) found that captopril decreased the iron concentration in serum shortly after administration; they suggested that an efficient mechanism that maintains a constant concentration of selected minerals may involve changes in the reabsorption of these minerals from the system blood to the tissues. In other studies, it was found that perindopril normalized increased urinary zinc excretion in hypertensive patients. In the present study we found that perindopril did not alter zinc status, in comparison with the control group. It should be appreciated that, in the treatment groups with supplementation, the significant increase in zinc and copper level was associated with a significant decrease in iron level in some tissues. The changes in mineral concentrations in tissues may be due to the interaction between iron and zinc and copper at the absorption and distribution stages of these minerals. However, it should be noted that the concentrations of minerals in the study groups (ID2 and AM2) were comparable to those in the control group. Therefore, the increase in zinc and copper and the decrease iron concentration in the tissues of the treatment groups receiving supplementation can be considered as a restoration of balance in mineral status and a reduction the disorders, as a result of treatment with indapamide and amlodipine. In conclusion, using the SHR model, we found that some hypotensive drugs, such as indapamide and amlodipine, lower
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zinc and copper levels in a tissue-dependent manner. These results suggest that antihypertensive treatment should include monitoring of minerals, and that patients on long-term therapy with hypotensive drugs may benefit from mineral supplementation. Clinical studies with hypertensive patients are required in order to examine the potential benefits of mineral supplementation in hypertension treatment.
Acknowledgments This research was supported by a grant from the National Science Center, Poland (2669/B/P01/2011/40).
References Arredondo, M., Nunez, M.T., 2005. Iron and copper metabolism. Mol. Asp. Med. 26, 313–327. Braun, L.A., Rosenfeldt, F., 2003. Pharmaco-nutrient interactions – a systematic review of zinc and antihypertensive therapy. Int. J. Clin. Pract. 67, 717–725. da Cunha, S., Albanesi Filho, F.M., da Cunha Bastos, V.L., Antelo, D.S., Souza, M.M., 2002. Thiamin, selenium, and copper levels in patients with idiopathic dilated cardiomyopathy taking diuretics. Arq. Bras. Cardiol. 79, 454–465. Ghayour-Mobarhan, M., Shapouri-Moghaddam, A., Azimi-Nezhad, M., Esmaeili, H., Parizadeh, S.M., Safarian, M., Kazemi-Bajestani, S.M., Khodaei, G.H., Hosseini, S. J., Parizadeh, S.M., Ferns, G.A., 2009. The relationship between established coronary risk factors and serum copper and zinc concentrations in a large Persian cohort. J. Trace Elem. Med. Biol. 23, 167–175. Hashemipour, M., Kelishadi, R., Shapouri, J., Sarrafzadegan, N., Amini, M., Tavakoli, N., Movahedian-Attar, N., Mirmoghtadaee, P., Poursafa, P., 2009. Effect of zinc supplementation on insulin resistance and components of the metabolic syndrome in prepubertal obese children. Hormones 8, 279–285. Joosten, M.M., Gansevoort, R.T., Mukamal, K.J., Kootstra-Ros, J.E., Feskens, E.J., Geleijnse, J.M., Navis, G., Bakker, S.J., 2013. PREVEND Study Group, 2013. Urinary magnesium excretion and risk of hypertension: the prevention of renal and vascular end-stage disease study. Hypertension 61, 1161–1167. Mahoney, J.J., Vreman, H.J., Stevenson, D.K., Van Kessel, A.L., 1993. Measurement of carboxyhemoglobin and total hemoglobin by five specialized spectrophotometers (Co-oximeters) in comparison with reference methods. Clin. Chem. 39, 1693–1700. Pikilidou, M.I., Lasaridis, A.N., Sarafidis, P.A., Tziolas, I.M., Zebekakis, P.E., Dombros, N.V., Giannoulis, E., 2007. Blood pressure and serum potassium levels in hypertensive patients receiving or not receiving antihypertensive treatment. Clin. Exp. Hypertens. 29, 563–573. Procedures for the Handling and Processing of Blood Specimens; Approved Guideline, Third edition. Reeves, P.G., O’Dell, B.L., 1986. Effects of dietary zinc deprivation on the activity of angiotensin-converting enzyme in serum of rats and guinea pigs. J. Nutr. 116, 128–134. Samaras, D., Samaras, N., Lang, P.O., Genton, L., Frangos, E., Pichard, C., 2013. Effects of widely used drugs on micronutrients: a story rarely told. Nutrition 29, 605–610. Skrzypczak, W., Dratwa-Chałupnik, A., Ozgo, M., Michałek, K., Lepczyński, A., Hejza, K., Siwa, J., 2010. Effect of converting enzyme inhibitor on copper and iron concentrations of blood plasma in calves during the neonatal period. Folia Biol. (Krakow) 58, 119–124. Suliburska, J., Bogdański, P., Pupek-Musialik, D., Krejpcio, Z., 2011. Dietary intake and serum and hair concentrations of minerals and their relationship with serum lipids and glucose levels in hypertensive and obese patients with insulin resistance. Biol. Trace Elem. Res. 139, 137–150. Swislocki, A.L., LaPier, T.L., Khuu, D.T., Fann, K.Y., Tait, M., Rodnick, K.J., 1999. Metabolic, hemodynamic, and cardiac effects of captopril in young, spontaneously hypertensive rats. Am. J. Hypertens. 12, 581–589. Thybo, N.K., Korsgaard, N., Eriksen, S., Christensen, K.L., Mulvany, M.J., 1994. Dosedependent effects of perindopril on blood pressure and small-artery structure. Hypertension 23, 659–666. Tubek, S., 2007. Role of zinc in regulation of arterial blood pressure and in the etiopathogenesis of arterial hypertension. Biol. Trace Elem. Res. 117, 39–51. Zuo, X.L., Chen, J.M., Zhou, X., Li, X.Z., Mei, G.Y., 2006. Levels of selenium, zinc, copper, and antioxidant enzyme activity in patients with leukemia. Biol. Trace Elem. Res. 114, 41–53.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
The influence of selected antihypertensive drugs on zinc, copper, and iron status in spontaneously hypertensive rats Joanna Suliburska a,n, Paweł Bogdanski b, Hieronim Jakubowski c,d,e a
Department of Human Nutrition and Hygiene, Poznan University of Life Sciences, Poznan, Poland Department of Internal Medicine, Metabolic Disorders and Hypertension, University of Medical Sciences, Poznan, Poland c Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, Poznan, Poland d Institute of Bioorganic Chemistry, Poznan, Poland e Department of Microbiology & Molecular Genetics, Rutgers University-New Jersey Medical School, International Center for Public Health, Newark, NJ, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 February 2014 Received in revised form 25 May 2014 Accepted 2 June 2014 Available online 11 June 2014
Mineral homeostasis in hypertensive patients may be affected by hypotensive drugs. The aim of this study was to assess the influence of selected antihypertensive drugs on mineral homeostasis in a rat model of hypertension. Eight-week-old male spontaneously hypertensive rats (SHRs) were treated with perindopril, metoprolol, indapamide, amlodipine, or no drug for 45 days. In another experiment, the SHRs were treated with indapamide or amlodipine in the presence of zinc and copper gluconate supplement. Lipids, glucose, and insulin levels along with superoxide dismutase and catalase activities were assayed in serum. Iron, zinc, and copper concentrations in serum, erythrocytes, and tissues were determined using the flame atomic absorption spectrometry. Blood pressure was measured using a tailcuff plethysmograph. Treatment with indapamide and amlodipine was found to significantly lower zinc levels in serum, erythrocytes, livers, and spleens of the SHRs, as well as copper levels in the kidneys, compared with the control no-drug group. A markedly higher concentration of glucose was found in the indapamide-treated rats. Supplementing the indapamide-treated SHRs with zinc and copper gluconate resulted in a significant decrease in both systolic and diastolic blood pressure, and also lowered serum glucose and triglyceride concentrations and HOMA (homeostasis model assessment-insulin resistance) values. The results show that indapamide and amlodipine disturb zinc and copper homeostasis in SHRs. Supplementation with zinc and copper restores mineral homeostasis in SHRs treated with indapamide and amlodipine, and also corrects metabolic imbalances while improving the antihypertensive efficiency of indapamide. & 2014 Elsevier B.V. All rights reserved.
Keywords: Copper Hypotensive drugs Iron Spontaneously hypertensive rats Zinc Chemical compounds studied in this article: Perindopril (PubChem CID: 107807) Amlodipine (PubChem CID: 2162) Indapamide (PubChem CID: 3702) Metoprolol (PubChem CID: 4171) Zinc gluconate (PubChem CID: 158040) Copper gluconate (PubChem CID: 10692)
1. Introduction Hypertension is a common disease in Western societies, and is usually treated with hypotensive drugs that are prescribed longterm. Antihypertensive drugs include angiotensin-converting enzyme inhibitors (ACEi), diuretics, calcium-channel blockers, beta blockers, and angiotensin receptor blockers (ARB); alpha blockers are also frequently used. These drugs are taken alone (monotherapy) or in combination (polytherapy). The long-term use of hypotensive drugs may cause side effects, such as inverse disturbances in electrolyte homeostasis, which is relatively common with the use of diuretics and ACEi (Braun and Rosenfeldt, 2003). n Correspondence to: Department of Human Nutrition and Hygiene, Poznan University of Life Sciences, ul. Wojska Polskiego 31, 60-624 Poznan, Poland. Tel.: þ 48 61 8487334; fax: þ48 61 8487332. E-mail address: [email protected] (J. Suliburska).
http://dx.doi.org/10.1016/j.ejphar.2014.06.003 0014-2999/& 2014 Elsevier B.V. All rights reserved.
In clinical and experimental studies, including our own, an association is observed between disordered sodium, potassium, magnesium, and calcium homeostasis and essential hypertension (Joosten et al., 2013; Pikilidou et al., 2007). It has been suggested that long-term antihypertensive therapy affects zinc pharmacokinetics, particularly in patients with conditions associated with disturbances of zinc homeostasis (Braun and Rosenfeldt, 2003). Antihypertensive therapy may significantly affect mineral status, and may require systematic monitoring (Pikilidou et al., 2007; Suliburska et al., 2011). Moreover, disorders in mineral status may impact lipid and glucose metabolism, and also mineral-dependent enzyme activity, such as that of superoxide dismutase (SOD) and catalase (CAT), in the body (Suliburska et al., 2011; Zuo et al., 2006). Drugs generally are capable of influencing the metabolism of trace elements in many ways from intestinal absorption to bioavailability and elimination. It is not known whether mineral supplementation or dietary modification might be an optimal
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treatment for hypertensive patients. However, in studies of mineral homeostasis in patients, the drugs used are rarely taken into consideration. Because the interactions between hypotensive drugs and nutrients are poorly understood, we have examined the effect of perindopril, metoprolol, indapamide, and amlodipine on iron, zinc, and copper homeostasis in the SHR model. We also studied the effect of the combined mineral supplements and drugs on mineral homeostasis in SHRs.
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Table 2 The amount of supplements in the diet in the experiment with supplementation. Group
Supplement (g/kg diet)
Total zinc Total copper (mg/kg) (mg/kg)
Zinc gluconiate Copper gluconiate C2 (n¼10) ID2 (n ¼10) AM2 (n ¼10)
– 0.627 0.627
– 0.129 0.129
70 160 160
13 31 31
C-control group, ID2-group with indapamide and zinc and copper supplements. AM2-group with amlodipine and zinc and copper supplements.
2. Materials and methods 2.1. Animals Eight-week-old male spontaneously hypertensive rats (SHRs) (Charles River Laboratories, Germany), derived from Wistar Kyoto rats with elevated blood pressure at the Kyoto School of Medicine, were used. The mean weight of the rats was 195 721 g. The animals were housed individually in stainless steel cages coated with metal-free enamel and kept under cycles of 12 h light and 12 h dark. The room temperature was maintained at 21 71 1C with 55–65% humidity. The animal procedures were approved by the local bioethics committee (approval no. 49/2009).
The second experiment (with supplementation) was designed on the basis of the first experiment. In this experiment drugs were used with potential influence on mineral status. Thirty animals were randomly assigned to three groups of 10 animals each: control diet (C2); diet with indapamide, zinc, and copper gluconate (ID2); and diet with amlodipine, zinc, and copper gluconate (AM2). The supplemented diets were prepared by mixing an appropriate proportion of the diet with the supplements (USP Merck). The proportions of the diet and supplements in each mix are shown in Table 2. The procedure of the second experiment followed that of the first. The experiments were begun following a 5-day period to allow the rats to adapt to laboratory conditions.
2.2. Experimental design
2.3. Blood-pressure measurements
Two experiments were performed. In the first (without supplementation), SHRs were randomly assigned to five groups of 10 animals each: the control, diet without drugs (C1), diet with perindopril (PR1), diet with metoprolol (MT1), diet with indapamide (ID1), and diet with amlodipine (AM1). The rats were fed a standard diet (maintenance diet for rats 1320, Altromin). The full composition of the diet is presented in Table 1. All rats were provided ad libitum diet and distilled water for 45 days. Dietary intake was recorded daily. Body weight was recorded each week prior to food distribution. In the diet of the noncontrol groups, perindopril, metoprolol, indapamide, and amlodipine were added at a rate of 0.2, 3.0, 0.03, and 0.2 mg/kg body mass of rat, respectively. The drug was administered in the diet, combined with chow, to which the rats had free access. Fresh solutions were prepared every day. The drug concentrations were adjusted so that the doses (calculated as milligrams per kilogram per day) were kept constant, regardless of diet intake and body weight. In this study, relatively low doses of these drugs were used.
Blood pressure was measured with a tail-cuff plethysmograph using a blood-pressure measuring system (MODEL MK-1030, Muromachi Kikai). The systolic and diastolic blood pressure was measured following 15 min warming at 37 1C in an animal holder made of dark brown acryl, allowing blood-pressure measurement under relatively stress-free conditions. An average of five readings was recorded for each animal. The rats were first habituated to the measurement device and remained unperturbed in the chamber throughout the inflation–deflation cycles.
Table 1 The composition of the base diet. Ingredient
Amount Ingredient
Total energy (kcal/kg) 2844 Total protein (% of energy) 24 Total fat (% of energy) 11 Total carbohydrate (% of energy) 65 Protein (g/100 g) 19 Fat (g/100 g) 4 Fiber (g/100 g) 6 Vitamin A (IU) 1500 Vitamin D3 (IU) 600 Vitamin B1 (mg/kg) 18 Vitamin B2 (mg/kg) 12 Vitamin B6 (mg/kg) 9 vitamin B12 (μg/kg) 24 vitamin C (mg/kg) 36 vitamin K3 (mg/kg) 3 Vitamin E (mg/kg) 75 Folic acid (mg/kg) 2
Amount
Biotin (μg/kg) 60 Nicotinic acid (mg/kg) 36 Pantothenic acid (mg/kg) 21 Choline chloride (mg/kg) 600 Calcium (g/kg) 9 Phosphor (g/kg) 7 Magnesium (g/kg) 3 Sodium (g/kg) 2 Potassium (g/kg) 1 Iron (mg/kg) 165 Manganium (mg/kg) 75 Zinc (mg/kg) 70 Copper (mg/kg) 13 Iodium (mg/kg) 1.5 Selenium (mg/kg) 0.6 Cobalt (mg/kg) 0.3
2.4. Tissue and serum collection The animals were fasted for 12 h prior to termination of the experiment. At the end of the experimental period, the animals were weighed and killed. The rats were anesthetized with a sodium thiopental injection (40 mg/kg body weight) and killed by cardiac puncture. The tissues (liver, spleen, kidneys, heart, pancreas, gonads) were dissected, weighed, and stored frozen (at 70 1C) for mineral content analysis. Each blood sample obtained by cardiac puncture was divided between a serum separator tube and a tube containing heparin sodium. The samples in the serum separator tubes were left undisturbed at room temperature for 30 min, and then centrifuged for 15 min at 1048 g at 4 1C; the supernatant was removed and stored at 70 1C. To isolate erythrocytes, whole blood in heparin sodium-containing tubes was centrifuged for 15 min at 1048 g at 4 1C, and the plasma was removed. Blood cells were washed thrice with 5 ml of 0.9% saline solution and centrifuged at 2493 g for 10 min at 4 1C. Following each centrifuging, saline solution was separated and the erythrocyte mass was placed in demineralized Eppendorf tubes and stored at 70 1C for minerals analysis. 2.5. Biochemical measurements Total cholesterol (TC), triglyceride (TG), and fasting glucose levels in serum were measured using commercial kits (Randox Laboratory Ltd, UK) at a diagnostic laboratory in Poznań, Poland. The concentration of cholesterol and triglycerides in serum was
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assayed using the enzymatic method. The concentration of glucose in the blood serum was estimated using the glucose oxidase method (Clinical and Laboratory Standards Institute/NCCLS, 2004). Serum insulin was determined with the radioimmunoassay method, using a rat insulin RIA kit (Insulin RIA Kit, Linco Research, USA). Insulin resistance was evaluated according to the homeostasis model assessment-insulin resistance (HOMA-IR) protocol: HOMA index ¼[fasting insulin (Um/l) fasting glucose (mmol/l)]/ 22.5. Hemoglobin was determined using the cyanmethemoglobin method (Mahoney et al., 1993). Serum superoxide dismutase (SOD) and catalase (CAT) activities were measured using an immunoenzymatic SOD and CAT activity assay kit (Cayman Chemicals, USA). 2.6. Determination of minerals The iron, zinc, and copper contents of the serum, erythrocytes, and tissues were determined following digestion in 65% (w/w) spectra pure HNO3 (Merck) in the Microwave Digestion System (MARS 5, CEM Corp., USA). Thereafter, the concentrations of iron, zinc, and copper in the mineral solutions were measured using the flame atomic absorption spectrometry method (AAS-3, Carl Zeiss, Jena, Germany). The mineral contents of serum and tissues were determined at the following wavelengths: for iron, 248.3 nm; for zinc, 213.9 nm; for copper, 324.8 nm. The accuracy of the method was verified with certified reference materials (bovine liver-trace elements, NIST-1577C, CERT and seronorm TRACE ELEMENTS Whole Blood L-2), and was 95–98% for iron, 95–96% for zinc, and 99–103% for copper. 2.7. Statistical analysis Detailed statistical analysis was performed using Statistica for Windows 10.0 (StatSoft, Poland). The results were expressed as arithmetic means with standard errors. One-way analysis of variance (ANOVA), followed by a post-hoc Turkey's test, were used to compare the data between groups. Student's t-test was used to compare the two means between groups from the first and the second experiment. The significance was set at the P o0.05 level.
3. Results The results of the first experiment, which did not involve supplementation, are shown in Tables 3–5. It was found that hypotensive treatment led to a decrease in both systolic (SBP) and diastolic blood pressure (DBP), but a
significant decrease was observed only in the groups given perindopril (SBP: 23.3%; DBP: 27.9%) and metoprolol (SBP: 19.7%; DBP: 30.9%), compared with the control no-drug group. Markedly higher concentrations of glucose in the ID1 group (þ11.5%), and markedly lower concentrations of triglycerides in the MT1 group ( 19.4%), were observed. Treatment with the diuretic indapamide or with the calcium-channel blocker amlodipine resulted in significant decreases in zinc in serum (ID1: 14.3%; AM1: 15.1%), erythrocytes (ID1: 7.0%; AM1: 5.0%), livers (ID1: 12.8%; AM1: 18.0%) and spleens (ID1: 9.0%; AM1: 10.2%) of the SHRs. Markedly lower liver copper ( 40.4%) and higher kidney iron ( þ15.0) in ID1 rats were observed. Perindopril markedly increased the concentration of iron in the rats' livers (þ 11.4%). Significantly lower concentrations of copper in the kidneys were found in the MT1 ( 13.3%), ID1 ( 17.8%), and AM1 groups ( 17.8%). The results of the second experiment, which employed supplementation, are shown in Tables 6–8. When the indapamidetreated (ID2) and amlodipine-treated (AM2) SHRs were supplemented with zinc and copper gluconate, significant decreases were observed in systolic (ID2: 19.4%; AM2: 20.0%) and diastolic blood pressure (ID2: 29.3%; AM2: 35.1%), while decreases were seen in serum glucose (ID2: 17.2%; AM2: 7.8%) and triglyceride (ID2: 30.0%; AM2: 25.0%) concentrations in the blood. The HOMA value for the ID2 group was significantly lower (ID2: 46.7%) than that of the C2 group. No significant differences were observed in tissue mineral concentrations between the study groups (ID2 and AM2) and the control group (C2). The results of the second experiment (with supplementation) were compared with the results of the first experiment (without supplementation). It was shown that indapamide combined with zinc and copper supplementation significantly decreased systolic and diastolic blood pressure (SBP: 14.5%, DBP: 15.9%), serum glucose ( 22.1%), triglycerides ( 22.2%) and HOMA ( 36.0%), and increased serum zinc level (17.6%) and zinc and copper concentration in erythrocytes (15.8% and 37.3% respectively), compared with ID1 group. In the group treated with indapamide and with supplementation, markedly higher concentrations of zinc and copper in the livers (9.3% and 31.1%), spleens (25.9%, 71.4%), and kidneys (39.9%, 32.4%) were found, as were markedly higher levels of zinc in the hearts (10.6%). In turn, the concentration of iron in livers, kidneys, hearts, and pancreases was significantly lower ( 19.7%, 21.4%, 25.8%, 14.9) in group ID2 than ID1. In the rats treated with amlodipine and with supplementation, significantly higher zinc concentrations in serum (18.8%) and erythrocytes (28.2%) were seen, compared to the rats without supplementation. Markedly higher concentrations of zinc and
Table 3 Blood pressure and biochemical parameters in rats in the experiment without supplementation. Parameter
SBP (mmHg) DBP (mmHg) Glucose (mmol/l) Insulin (pmol/l) HOMA Cholesterol (mmol/l) Triglyceride (mmol/l) SOD (U/ml) CAT (nmol/min/ml)
Groups C1 (n¼10)
PR1 (n¼10)
MT1 (n¼ 10)
ID1 (n ¼10)
AM1 (n ¼10)
197.2 7 27.7 123.9 7 15.7 6.17 0.6 132.8 7 41.4 5.03 7 1.53 1.127 0.20 0.36 7 0.01 6.92 7 0.85 44147 1040
151.3 716.4a 89.3 725.7a 6.5 70.9 139.5 753.3 5.63 72.42 1.17 70.19 0.39 70.03 6.15 71.20 3951 7808
158.3 7 31.3a 85.6 7 29.4a 5.9 7 1.0 124.07 39.6 4.34 7 1.58 1.017 0.13 0.29 7 0.05a 6.55 7 0.80 36067 912
180.2 720.7 101.1 719.8 6.8 70.6a 107.0 720.7 4.46 70.89 1.05 70.14 0.36 70.07 6.72 71.06 3371 7950
170.27 30.4 103.6 7 26.9 6.17 0.6 136.3 7 50.3 5.147 1.80 1.047 0.13 0.34 7 0.07 6.83 7 0.89 4898 7 1055
C1-control group, PR1-group with perindopril, MT1-group with metoprolol, ID1-group with indapamide, AM1-group with amlodipine, SBP-systolic blood pressure, DBP-diastolic blood pressure, HOMA-insulin resistance index, SOD-superoxide dismutase, CAT-catalase. a
P o0.05 compared to C1.
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Table 4 Minerals concentration in serum and erythrocytes in the experiment without supplementation. Parameter
Groups C1 (n¼ 10)
PR1 (n¼ 10)
MT1 (n¼10)
ID1 (n ¼10)
AM1 (n ¼10)
Serum (μmol/l) Iron Zinc Copper
23.3 7 1.1 11.9 7 0.6 13.3 7 0.6
23.4 7 1.0 12.0 7 0.6 13.2 7 0.6
23.9 71.1 12.1 70.6 13.0 70.6
24.17 1.2 10.2 7 0.5a 12.6 7 0.7
23.9 7 1.2 10.17 0.6a 12.8 7 0.6
Erythrocytes Iron (μmol/g Hb) Zinc (μmol/g Hb) Copper (nmol/g Hb)
41.9 7 2.0 0.417 0.03 40.0 7 3.3
44.6 7 2.2 0.40 7 0.02 41.2 7 5.2
46.1 72.3 0.43 70.09 44.1 74.1
45.7 7 2.3 0.38 7 0.03a 36.2 7 2.2b
45.17 2.7 0.39 7 0.06a 39.3 7 3.8
C1-control group, PR1-group with perindopril, MT1-group with metoprolol, ID1-group with indapamide, AM1-group with amlodipine. a b
P o0.05 compared to C1. Po 0.05 compared to MT1.
Table 5 Iron, zinc and copper concentration in tissues in the experiment without supplementation (μg/g wet wt.). Parameter
Groups C1 (n¼10)
PR 1(n ¼10)
MT 1(n¼ 10)
ID1 (n¼ 10)
AM 1(n¼ 10)
Liver Iron Zinc Copper
158.6 7 9.1 40.5 7 1.5 5.2 7 0.2
176.7 7 10.3a 41.0 7 2.4 4.8 7 0.3
164.2 75.4 40.1 72.2 4.8 70.3
165.2 7 6.8 35.3 7 1.5a 3.17 0.4a
167.7 7 13.6 33.27 1.7a 5.17 0.4
Spleen Iron Zinc Copper
727.0 7 96.8 25.5 7 2.6 1.8 7 0.6
737.9 7 105.8 24.27 1.3 1.4 7 0.6
661.2 7103.1 24.371.0 1.7 70.4
664.6 7 105.6 23.2 7 1.0a 1.4 7 0.5
653.4 7 97.7 22.9 7 0.9a 1.4 7 0.4
Kidney Iron Zinc Copper
61.4 7 6.9 20.0 7 1.1 4.5 7 0.1
68.57 8.0 20.4 7 0.5 4.17 0.2
68.8 73.8 20.6 71.1 3.9 70.2a
70.6 7 5.4a 20.3 7 0.8 3.7 7 0.3a
65.2 7 5.3 20.6 7 1.2 3.7 7 0.3a
Heart Iron Zinc Copper
83.17 7.0 26.8 7 1.5 5.7 7 0.2
91.7 7 14.2 26.5 7 2.6 6.0 7 0.7
85.7 712.3 26.2 71.7 5.8 70.6
90.6 7 6.9 25.4 7 1.6 5.5 7 0.3
81.0 7 10.1 25.4 7 1.7 5.4 7 0.5
Pancreas Iron Zinc Copper
25.17 2.8 26.9 7 1.5 1.17 0.2
27.0 7 3.3 26.7 7 2.3 1.2 7 0.3
27.2 73.5 28.6 74.1 1.2 70.2
28.17 2.4 29.8 7 3.9 1.17 0.2
26.7 7 1.8 28.4 7 4.1 1.3 7 0.2
Gonads Iron Zinc Copper
22.9 7 1.4 30.8 7 2.2 1.9 7 0.2
24.47 1.0 32.4 7 1.2 1.8 7 0.2
22.5 71.6 31.1 72.7 1.8 70.1
23.5 7 1.8 29.5 7 0.9 1.7 7 0.2
23.3 7 2.2 30.0 7 1.0 1.6 7 0.2
C1-control group, PR1-group with perindopril, MT1-group with metoprolol, ID1-group with indapamide, AM1-group with amlodipine. a
P o0.05 compared to C1.
copper were also observed in the kidneys (31.1%, 24.3%) and hearts (15.4%, 9.4%) of in group AM2, compared with those of AM1. Moreover, zinc concentration in the livers (20.5%) and spleens (25.8%) was markedly higher in the AM2 group than in AM1. Treatment with amlodipine and supplementation decreased iron levels in the livers ( 18.7%), kidneys ( 13.8%), and pancreases ( 13.9%).
4. Discussion The influence of indapamide and amlodipine on zinc and copper homeostasis in SHRs is a new finding of this study. Furthermore, to the best of our knowledge, this study is the first experimental demonstration of a positive outcome on metabolic and mineral homeostasis in SHRs associated with zinc and copper supplementation during indapamide therapy.
Our results suggest that hypotensive drugs disturb zinc and, to a lesser extent, copper homeostasis in SHRs. Decreases in zinc levels in serum, erythrocytes, the liver, and the spleen during the ID and AM treatments were observed in the absence of any significant decrease in blood pressure in the ID-treaded and AMtreated animals. The decrease in zinc was accompanied by an increase in glucose serum levels in the ID-treated SHRs. Moreover, our results suggest that a combination of indapamide or amlodipine therapy with zinc and copper supplementation restores mineral homeostasis. In the case of indapamide, there is also a positive effect on lipid metabolism, carbohydrate metabolism, and blood pressure. The results obtained in the present study suggest that the interaction between zinc and hypotensive drugs exerts a pharmacokinetic effect, and may be caused by the inhibition of renal zinc reabsorption, resulting in zincuria (Braun and Rosenfeldt, 2003; Samaras et al., 2013). It is possible that antihypertensive drugs
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Table 6 Blood pressure and biochemical parameters in rats in the experiment with supplementation. Parameter
Groups C2 (n¼ 10)
SBP (mmHg) DBP (mmHg) Glucose (mmol/l) Insulin (pmol/l) HOMA Cholesterol (mmol/l) Triglyceride (mmol/l) SOD (U/ml) CAT (nmol/min/ml)
191.1 719.0 120.3 713.8 6.4 70.2 129.1 751.6 5.29 72.30 1.25 70.09 0.40 70.05 6.85 70.81 4448 71050
Parameter ID2 (n ¼10)
AM 2(n ¼10) a,A
154.0 7 25.2 85.0 7 16.7a,A 5.3 7 0.1a,A 86.1 7 49.9 2.85 7 1.56a,A 1.357 0.28 0.28 7 0.01a,A 6.94 7 0.80 3860 7 866
a
152.9 7 24.7 78.17 25.7a 5.9 7 0.4a 120.357 48.2 4.36 7 1.43 1.36 7 0.14 0.30 7 0.06a 6.98 7 0.86 43507 982
C2-control group, ID2-group with indapamide with Zn and Cu supplements. AM2-group with amlodipine with Zn and Cu supplements, SBP-systolic blood pressure. DBP-diastolic blood pressure, HOMA-insulin resistance index, SOD-superoxide dismutase, CAT-catalase. a
P o0.05 compared to C2. A Po 0.05 compared to ID1.
Table 7 Minerals concentration in serum and erythrocytes in the experiment with supplementation. Parameter
Groups C2 (n ¼10)
ID2(n¼ 10)
AM2 (n¼10)
Serum (μmol/l) Iron Zinc Copper
23.5 7 1.1 11.8 7 0.61 13.2 7 0.6
23.2 7 1.1 12.0 7 0.6A 13.0 7 0.6
23.9 71.2 12.0 70.6B 13.2 70.6
Erythrocytes Iron (μmol/g Hb) Zinc (μmol/g Hb) Copper (nmol/g Hb)
45.7 7 5.7 0.46 7 0.11 40.6 7 3.9
47.1 7 4.2 0.44 7 0.11A 49.7 7 4.9A
45.8 72.6 0.50 70.16B 33.9 74.8a
C2-control group, ID2-group with indapamide with Zn and Cu supplements. AM2-group with amlodipine with Zn and Cu supplements. a A B
Table 8 Minerals concentration in tissues in the experiment with supplementation (μg/g wet wt.).
P o0.05 compared to ID2. Po 0.05 compared to ID1. Po 0.05 compared to AM1.
affect zinc reabsorption in the kidney by decreasing renal Na/H antiporter activity. Interestingly, angiotensin II is a potent inducer of Na/H antiporter, and the synthesis of this hormone depends on the activity of angiotensin-converting enzyme (ACE) (Reeves and O’Dell, 1986). It is known that thiazides inhibit NaCl transport in the distal tubule, and may affect zinc reabsorption through this mechanism (Braun and Rosenfeldt, 2003; Samaras et al., 2013). Indapamide is a thiazide-like diuretic. In some human studies, it has been found that thiazide diuretics may increase zinc urinary excretion; in some patients, decreased serum zinc levels in serum have also been observed (Braun and Rosenfeldt, 2003). The results of clinical studies have revealed zinc disturbances caused by commonly prescribed diuretics. Increases in urinary zinc and decreases in both plasma and erythrocyte zinc levels were seen. Although, in the present study, urinary zinc was not assayed, the decreased levels of zinc in the analyzed rat tissues were most likely caused by the loss via urinary excretion. This is supported by the fact that no accumulation of zinc was observed in any of the analyzed tissue following treatment with indapamide and amlodipine. Our results show the relation between zinc homeostasis and blood pressure. It is suggested that disturbances in zinc homeostasis may affect blood pressure. Tubek (2007) indicated that low zinc concentrations in the extracellular space lead to less efficient
Groups C2 (n¼ 10)
ID2 (n¼ 10)
AM2 (n¼ 10)
Liver Iron Zinc Copper
135.879.3 38.7 70.7 4.2 70.2
132.7 7 11.2A 38.6 7 2.4A 4.5 7 0.7A
136.3 7 18.1B 40.0 7 2.1B 4.5 7 0.4
Spleen Iron Zinc Copper
638.3 777.8 28.4 71.2 2.2 70.4
631.3 7 94.7 29.2 7 2.0A 2.4 7 0.8A
635.97 43.7 28.8 7 0.8B 1.9 7 0.4
Kidney Iron Zinc Copper
56.8 76.2 28.3 71.2 4.7 70.3
55.5 7 2.4A 28.4 7 1.7A 4.9 7 0.5A
56.2 7 7.5B 27.0 7 1.2B 4.6 7 0.2B
Heart Iron Zinc Copper
69.2 75.7 27.7 72.1 5.8 70.3
67.2 7 6.9A 28.17 1.4A 5.8 7 0.6
70.27 9.0 29.3 7 2.5B 5.9 7 0.3B
Pancreas Iron Zinc Copper
23.4 74.3 26.4 73.4 1.3 70.3
23.9 7 0.7A 25.9 7 2.0 1.3 7 0.2
23.0 7 1.5B 26.6 7 2.5 1.2 7 0.3
Gonads Iron Zinc Copper
20.1 71.6 28.0 70.6 1.6 70.1
20.2 7 0.9A 28.5 7 1.0 1.7 7 0.2
20.7 7 2.6 29.0 7 1.2 1.7 7 0.3
C2-control group, ID2-group with indapamide with Zn and Cu supplements. AM2-group with amlodipine with Zn and Cu supplements. A B
Po 0.05 compared to ID1. P o0.05 compared to AM1.
blocking of calcium channels, favoring the movement of calcium into the cells and its accumulation there, leading to increased blood pressure. In our study, the lower zinc tissue concentration in the groups treated with indapamide and amlodipine was associated with slightly decreased blood pressure, while in the groups with perindopril and metoprolol (which lacked disturbed zinc homeostasis), the blood pressure was reduced. Moreover, the restoration of zinc content through diet supplementation caused a decrease in blood pressure in rats that was not observed in the nonsupplemented animals. The disorder in copper homeostasis that was observed in the SHRs following treatment with indapamide and amlodipine may occur because of metabolic interrelationships between zinc, copper, and iron. The decrease in the zinc level probably affected iron homeostasis (manifested as a slight increase in the liver and kidney iron levels), and the change in the concentration of these minerals may be related to the change in the tissue copper level (Arredondo and Nunez, 2005). The concentration of copper in serum and erythrocytes was not changed by hypotensive drugs, which is consistent with the results of clinical trials (da Cunha et al., 2002). In this study, metoprolol also influenced copper levels in the kidneys of rats. In some studies, it has been found that antihypertensive drugs disturb homeostasis of electrolytes and other minerals through alterations in kidney or intestine mineral reabsorption, as well as through their chelating properties and by causing changes in the reabsorption of minerals from the system blood to the tissues (Braun and Rosenfeldt, 2003; Samaras et al., 2013). The mechanism involved in the change in kidney copper concentration as a result of metoprolol treatment may be similar. The decrease in copper concentration in the kidney was the only change in the mineral status of rats treated with metoprolol, and
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this change was associated with a decrease in the triglyceride level in serum. These results confirm the positive correlation between copper and triglycerides observed in other studies (Suliburska et al., 2011). It should be noted that disorders in zinc and copper homeostasis caused by hypotensive drugs may produce a wide range of negative effects in the body. It is known that zinc deficiency may predispose to glucose intolerance and insulin resistance, while copper deficiency leads to hypercholesterolemia and impaired endothelium-dependent arterial relaxation (Ghayour-Mobarhan et al., 2009). In the present study, we found that the lower status of zinc in SHRs treated with indapamide was associated with higher serum glucose concentrations. This could be caused by a decrease in the tolerance of glucose, due to the lower level of zinc in the rats, or to a lower level of insulin in serum, or both. It is known that zinc plays a major role in the synthesis and action of insulin. Zinc is a component of many enzymes, and is involved in the synthesis, storage, and release of insulin (Hashemipour et al., 2009). In the ID1 group, a slightly lower level of insulin was observed than in the control group. The supplementation of the diet with zinc and copper in SHRs treated with indapamide resulted in a significant reduction in triglycerides, glucose, and blood pressure. The combination of this drug with zinc and copper supplements increased the effectiveness of the treatment, and reduced the side effects of the drug. The significant decrease in the blood pressure of the rats treated with both indapamide and zinc and copper supplementation may be caused by the increase in SOD activity (although this increase did not reach statistical significance). In this study, we also investigated perindopril and observed a significantly higher iron concentration in the liver of rats on this drug. Similar to captopril (the first ACE inhibitor approved for use in patients), perindopril reduces blood pressure without adverse effects on serum glucose, insulin, or lipids in SHRs (Swislocki et al., 1999). Several studies have shown that, during treatment, perindopril had a persistent dose-dependent effect on blood pressure (Thybo et al., 1994). Skrzypczak et al. (2010) found that captopril decreased the iron concentration in serum shortly after administration; they suggested that an efficient mechanism that maintains a constant concentration of selected minerals may involve changes in the reabsorption of these minerals from the system blood to the tissues. In other studies, it was found that perindopril normalized increased urinary zinc excretion in hypertensive patients. In the present study we found that perindopril did not alter zinc status, in comparison with the control group. It should be appreciated that, in the treatment groups with supplementation, the significant increase in zinc and copper level was associated with a significant decrease in iron level in some tissues. The changes in mineral concentrations in tissues may be due to the interaction between iron and zinc and copper at the absorption and distribution stages of these minerals. However, it should be noted that the concentrations of minerals in the study groups (ID2 and AM2) were comparable to those in the control group. Therefore, the increase in zinc and copper and the decrease iron concentration in the tissues of the treatment groups receiving supplementation can be considered as a restoration of balance in mineral status and a reduction the disorders, as a result of treatment with indapamide and amlodipine. In conclusion, using the SHR model, we found that some hypotensive drugs, such as indapamide and amlodipine, lower
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zinc and copper levels in a tissue-dependent manner. These results suggest that antihypertensive treatment should include monitoring of minerals, and that patients on long-term therapy with hypotensive drugs may benefit from mineral supplementation. Clinical studies with hypertensive patients are required in order to examine the potential benefits of mineral supplementation in hypertension treatment.
Acknowledgments This research was supported by a grant from the National Science Center, Poland (2669/B/P01/2011/40).
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