Fluctuation of Zinc, Copper, Magnesium and Calcium Concentrations in Guinea Pig Tissues after Administration of Captopril (SQ 14225)

Journal of

Trace Elements

J. Trace Elements Med. BioI. Vol. II, pp. 32-36 (1997)

In Medicine and Biology

© 1997 by Gustav Fischer Verlag

Fluctuation of Zinc, Copper, Magnesium and Calcium Concentrations in Guinea Pig Tissues after Administration of Captopril (SO 14225) v.-P. KOTSAKI-KOVATSI, G. KOEHLER-SAMOUILIDIS*, A. KOVATSIS* and G. ROZOS* Laboratory of Phannacology and *Laboratory of Biochemistry and Toxicology, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki 540 06, Macedonia, Greece (Received April/December 1996)

Summary

The effect of the administration of captopril on Zn (zinc), Cu (copper), Ca (calcium) and Mg (magnesium) concentrations in guinea pig tissues was studied. For nine weeks 2 mg captopril per kg b.w. were administered daily to adult male guinea pigs intraperitoneally. The concentrations of the studied metals were detennined in several tissues. Captopril significantly decreased Zn concentration in liver, Cu concentration in liver, adrenals. jejunum. urine and hair and Mg concentrations in blood and urine. A significant increase was observed in testicular and epididymal Zn, in heart. epididymal and fecal Cu, in Mg concentration of lung, kidney, adrenals, jejunum, epididymis and hair and in Ca concentrations in brain, heart, lung, kidney, spleen and stomach. No significant changes were observed in the colon and the thigh bone concentrations of the various elements tested. In conclusion Captopril treatment can produce translocation and/ or elimination of Zn, Cu, Mg and Ca ions in various tissues of guinea pigs. Keywords: Captopril, metal translocation, guinea pigs.

Introduction

The mineral elements play varIOUS roles in cell growth, differentiation. and general metabolism, and in the immune system. Unlike some other nutrients, they do not provide energy for body functions. Instead, after participating in several regulatory functions in tissues, they are discharged. Hence, their regular supply in the diet is essential to maintain a constant turnover. Minerals such as calcium, magnesium, potassium, sodium, phosphorus, chloride and sulphur are needed in large quantities as they participate in body structure, while iron, cop-

Reprint requests to: Assoc. Prof. Dr. V-Po Kotsaki-Kovatsi, Laboratory of Pharmacology, Faculty of Veterinary Medicine, Aristotle University ofThessaloniki, Thessaloniki 54006, Macedonia, Greece.

per, fluoride, iodine, molybdenum, manganese, chromium, selenium, and zinc are needed in traces. However, if the essential elements are taken in large quantities, they become dangerous for the organism (I) . There are several reports of drugs binding competitively with the essential elements. These drugs form complexes with divalent cations in the organism (2,3) and in this way biologically important metal ions can be displaced or excreted from the organism. The results of recent experiments showed that translocation and/or elimination of Zn, Cu, Mg and Ca took place when drugs capable of forming complex compounds with metal ions were administered to test animals (4,5), which explained, many of the observed adverse effects of these drugs. Captopril, 1- [2 (S) - 3-mercapto 2-methylpropionyl]-L- proline, SQ 14225, an active inhibitor of angiotensin converting enzyme (ACE), is used

Metal concentrations in guinea pig tissues after administration of captopril

for the treatment of congestive heart failure and is well known as an effective antihypertensive agent (6,7,8,9). Results of recent research have shown that captopril complexes with Cu (II) and Zn (II) in vitro (10) and we believe that the same thing may also happen in vivo. Christie et al (6) studied the binding of metal ions by captopril in vitro and assessed the ability of this drug to mobilise Cu in vivo from blood plasma. Jay et al (10) suggested that the main mechanism by which captopril inhibits the superoxide anion-mediated production of phenyl radicals is the direct interaction of the drug with the metals that catalyze the autoxidation process and particularly with Cu 2+. This may have as a result the translocation and/or elimination of these metals from the organism and may explain side effects which are often associated with metal (Zn, Cu, Mg, Ca) depletion in the organism. The deficiency of Zn, which is important for a variety of Zn dependent enzymes, can lead to growth retardation, hypogonadism, anorexia, acrodermatitis, impaired wound healing and impaired immunity ( 11 ). Magnesium's role in enzymatic reactions has long been recognized (12,13 ). More than 260 enzymes have been found to require Mg2+ for their activation (14). Among these are the ATPases, which provide the basic energy for cell functioning (11). Their deficiency leads to a secondary deficiency of Ca and reduction in bone mass. The formation of Cu and Zn complexes with Captopril in vitro led us to the hypothesis that the same could happen in vivo with both Cu and Zn as well as with other metal ions. The result of this complex formation could be redistribution and/or excretion of these metal ions. Selection of the specific metals for analysis was based upon the essential need of these metals for life and their ability, from the chemical point of view, to form complexes with captopril. The aim of the present study was to investigate the effect of captopril on the concentrations of Zn, Cu, Mg and Ca in guinea pig tissues.

33

captopril solution to be injected i.p. The administered dose was 2 mg per kg b.w. The animals were given one injection every day for nine weeks. The vehicle was injected i.p. in six control animals. Two days after the last injection the animals were sacrificed by exposure to diethyl ether vapours. The day prior to sacrificing, the animals were put into metabolic cages in order to collect 24 h urine and feces. Urine and feces were collected in glass tubes and stored at 20°C until analysis. Immediately after death, blood was collected directly from the heart using heparinized syringes. Brain, heart, lung, liver, kidney, adrenals, spleen, a portion of the stomach, jejunum and colon, testis, epididymis, thigh bone and about 2 g of hair from the neck were removed. The tissues were blotted dry, weighed and kept frozen (20°C) until further analysis. Wet digestion with concentrated nitric and perchloric acids (I: I) was used and digests were analysed for zinc, copper, calcium and magnesium by flame atomic absorption spectrophotometry in air/acetylene flame at 214 nm, 324 nm, 211 nm and 285 nm wavelength, respectively (8). A calibration curve was constructed using Cu, Zn, Ca and Mg standard solutions (l,000±0,002 g/l, Merck, Darmstadt, Germany) diluted in destilled water at concentrations of 0,5 mg/L, 1,0 mg/L and 2 mg/L for Cu, of 0, I mg/L, 0,5 mg/L and 1 mg/L for Zn, of 0,5 mg/L, 1,0 mg/L, 2,5 mg/L and 5 mg/L for Ca and of 0, I mg/L, 0,25 mg/L and 0,5 mg/L for Mg. All curves were linear. According to Gorsuch (15) wet digestion results in element recoveries between 99-100 %. Determination of element concentrations was calculated from a standard curve obtained by plotting absorption against Ilg/ml of the element: ppm element =[(Ilg element/ml from curve)x(dilution factor, ml»)/g sample. Student's t-test was used for statistical analysis with p < 0.05 being considered significant.

Results and Discussion Material and Methods

Fifteen (nine test animals and six controls) male Hartley-Albino guinea pigs, twelve months old and 750-950 g in weight were used. They were housed in boxes at 20noc with lighting conditions of 12 h of light and 12 h of darkness. The animals were provided daily ad libitum with water and a balanced diet. (The metal content of the diet was as follows: 1,2 g!kg of Ca, 50 mg/kg of Zn, 7 mg!kg of Cu, 50 mg!kg of Mg). An aqueous solution of captopril 2 mg/ml was prepared before each injection. The animals were weighed before each injection in order to calculate the volume of

During our study we observed no clinical signs in the test and control animals. The consumed diet of both groups was 39 g pellets per kg b. w./daily at the begin and the end of the study. Also there were no changes in the b. w. of both groups. Post mortem sections revealed no lesIons. When considering the effect of captopril on the concentrations of Zn, Cu, Mg and Ca in the examined tissues it is necessary to focus our attention on its ability to form complexes with the above mentioned ions. It is known that metals are associated with a variety of proteins and other micro- and macro- molecules (16).

34

Y.-P. Kotsaki-Kovatsi, G, Koehler-Samouilidis, A. Kovatsis and G. Rozas

It is also evident from earlier studies that excess of any one of the elements in the cell may alter the concentrations of the others (17). It has been proved that one of the most important characteristics of captopril, which forms the basis of its high affinity for angiotensin-converting enzyme (ACE), is the presence of a sulfhydryl group that complexes the zinc ion of the enzyme (l0). It is also known that several metals have a great affinity for the sulfhydryl group. Membrane proteins containing SH groups therefore are helpful in metal transport. In Tables 1 and 2 we present the results of captopril administration on the Zn, Cu, Mg and Ca concentrations in the examined tissues, blood, urine, feces and hair, Liver Zn, Cu concentrations in the liver, adrenals, jejunum, urine and hair, and blood and urine Mg concentrations were decreased (p<0.05). A significant increase was observed in testicular and epididymal Zn, in Cu concentrations in heart, epididymis and feces, in Mg concentrations in lung, kidney, adrenals,

jejunum, epididymis and hair and in Ca concentrations in brain, heart, lung, kidney, spleen and stomach (p0.05) were observed in the colon and thigh bone concentrations of the ions study. The elimination of these essential elements from various vital organs not only disturbs the physiological, metabolic and regulatory functions in the cells, but it may also lead to several disorders. Mg and Zn are important constituents of enzymes and proteins. Mg is also an important regulator of Na and Ca transport in arterial smooth muscle, and its deficiency causes an increase in the contractility of arteries (l). Cu is an important constituent of the tissues of the circulatory system and of many enzyme and co-enzyme systems. Decreased levels of essential elements during captopril administration seem to have a direct effect on various macromolecules in different tissues. For example, Zn-dependent enzymes, such as alkaline phosphatase and lactic dehydrogenase, are significantly inhibited (18,19) during periods of decreased concentration of this essential element.

Table L Influence of captopril injected i.p. on zinc, copper, magnesium and calcium concentrations in guinea pig tissues -------

Zn Organs

BRAIN

HEART

LUNG

LIVER

KIDNEY

AORENAL

SPLEEN

STOMACH

JEJUNUM

Cu

Control" -

Test"

x SO P

19.93 2.34

x SO P

26.32 6.51

x SO P

17.27 4.66

x SO P

56.52 4.21

x SO P

25.65 3.47

x SO P

16.67 3.52

x SO P

26.14 4.71

x SO P

20.45 9.45

x SO P

36.17 12.58

20.07 2.42

Mg

Control'

--------------

6.13 1.47

6.06 1.29

8.14 3.16

NS

Control' IX3.49 13.13

11.92 2.45

236.62 11.86

5.9 2.23

6.54 2.17

28.04 4.61

+

16.17 4.7

NS

NS 25.58 4.76

16.69 6.78

NS

15.94 6.36

39.64 10.63

10.1 3.8

151.51 22.2

+

---"-------

a: Number of animals: control (6), test (9); +: p<0.05, N.S.: Not significant

145.31 17.66

13.0 4.71

22.3 3.54 +

168.0 14.42 +

15.41 4.64 +

NS 24.78 3.81

12.63 4.12 NS

293.79 22.87

132.89 20.0

289.27 41.11

11.8 1.17

NS

NS 25.93 5.77

168.68 1.93 +

155.04 19.32

333.33 49.43

6.44 3.02

6.55 2.92

+

11.66 2.87

32.38 6.74

NS 245.57 25.68

116.03 20.87

+

NS

14.31 1.27 +

229.71 21.50

201.88 10.89

14.79 1.91 +

+

11.6 2.74

11.44 3.72

5.81 0.45

NS 13.87 Ul9

56.69 9.97 +

149.19 15.95

242.45 44.27

NS

26.97 5.71

45.74 6.24

Test"

- - - - _..- -

+

20.99 3.05

13.72 1.92

NS 22.1 10.95

189.37 17.35

267.23 34.93

126.26 14.63

+

22.82 5.13

Control'

NS

NS 41.73 8.97

Test'

NS

+

NS

NS

Test'

NS

23.09 7.26

wet tissue)

Ca

--

NS 28.64 11.28

(~g/g

--------

-

23.23 4.29

20.5 6.83 NS

Metal concentrations in guinea pig tissues after administration of captopril Table 2. Influence of captopril injected i.p. on zinc, copper, magnesium and calcium concentrations in guinea pig tissues Zn

TESTIS

EPIDIDYMIS

BONE

BLOOD

URINE

FECES

HAIR

wet tissue)

Ca

Control"

Test"

Control'

Test"

Control"

Test"

Control"

Test"

x SD P

30.61 3.07

37.06 7.45

8.91 4.4

6.56 1.61

201.14 48.0

242.16 44.49

18.64 6.52

21.38 5.72

x SD P

12.8 0.38

x SD P

27.91 3.71

x SD P

242.19 54.7

x SD P

9.24 2.08

x SD P

22.23 4.4

x SD P

248.0 18.95

x SD P

302.36 14.28

Organs

COLON

Mg

Cu

(~g/g

NS

NS 19.43 6.65

5.8 1.18

36.84 3.96

17.04 1.58

+

24.19 3.16

NS

NS

3109.46 194.76

NS

+ 322.43 48.81

102.35 4.91

NS

141.55 30.57

NS

33.53 13.33 NS

*21.69 5.22

*74.31 14.15

1360.67 580.24

*75.17 22.15 NS

1562.9 485.21 NS

21.52 2.74

26.69 1.94

26.96 10.29

+

+ 367.7 77.31

NS 54.31 17.53

*28.65 4.79

85.07 8.04

*69.51 21.87

+ 23.16 2.54

27.9 2.9

11.56 7.99 NS

3073.8 256.99

72.94 13.89

NS 18.88 2.87

14.31 4.64

NS 1.12 0.39

1.57 0.43

115.37 12.19

92.48 6.76

NS 9.18 1.82

NS

+ 6.06 1.08

6.08 0.79

9.52 2.35

8.8 2.28

NS

+ 294.9 70.33

158.51 12.40

171.96 12.26

NS

+

NS

NS 6.0 0.67

35

207.54 49.56

+

265.73 49.56

302.36 14.28

+

367.7 77.31 NS

a: Number of animals: control (6), test (9); +: p<0.05, N.S.: Not significant; *: mg/g wet tissue resp. mg/ml; 0: mg/L

Clinical trials have shown that side effects are sometimes associated with captopril (20), especially in patients receiving high doses (>450 mg per day). These side effects include skin rashes, dysgeusia and neutropenia, clinical signs often associated with Zn and/or Cu depletion. Captopril also causes disorders in the reproductive system. AI Shabanah et al (21) report a significantly reduced mean number of implantations, a retardation of intrauterine growth of baby rats and reduced viability of newborns. During our studies we observed an increase of Zn, Cu, and Mg concentrations in the epididymis and an increase of Zn concentrations in the testis. These results support our opinion (4,5) that drugs capable of forming complexes with metal ions can cause translocation and/or elimination of these ions from the organism. In conclusion, the treatment of guinea pigs with captopril can produce translocation and/or elimination of Zn, Cu, Mg and Ca ions in various tissues.

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