Oxidase Activity of Ceruloplasmin and Concentrations of Copper and Zinc in Serum in Chronic Arterial Occlusion of the Lower Limbs

Jourflal of

Trace Elements

J. Trace Elements Med. BioI. Vol. 13, pp. 76-81(1999)

In Medicine and Biology

© 1999 Urban & Fischer

Clinical Study

Oxidase Activity of Ceruloplasmin and Concentrations of Copper and Zinc in Serum in Chronic Arterial Occlusion of the Lower Limbs M. ISKRA and W. MAJEWSKI* Department of General Chemistry, *Department of General and Vascular Surgery, Karol Marcinkowski University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland (Received June/October 1998)

Summary The presence of ischaemic tissue excites an inflammatory reaction and synthesis of acute phase proteins (APhPs). Ceruloplasmin (Cp) protein binds 90% of the copper in plasma and it is one of the positive APhPs, and its concentration increases in infection, inflammation or necrosis. The study presents the relationship of the oxidase activity of Cp and concentrations of Cu and Zn in serum of men with different degrees of ischaemia of the lower limbs. The subjects were 32 men with chronic arterial occlusion (AO) of the lower limbs. The oxidase activity of Cp was measured in serum with o-dianisidine as a substrate. Concentrations of Cu and Zn were determined by using atomic absorption spectrometry. The mean activity of Cp in serum in AO (I 73±69.2 VII) was higher as compared with the control group (l23.7±28.6 UII), and in men with critical ischaemia (~194.8 VII) than in men with a moderate level of ischaemia (l09.3±31.6 UII). The mean concentrations of eu and Zn in serum were found to be higher in AO (22.2±4.2 and 19.1±6.9 f..tmolll, respectively) than in the control group (I6.3±1.8 and 15.2±2.3 f..tmolll), and in men with critical ischaemia (~22.2 and 19.1 f..tmolll) than in men with a moderate level of ischaemia (I8.5±3.3 and 14.5±4.3 f..tmolll). Significant positive correlation coefficients were calculated for the activities of Cp and concentrations of Cu in the control group (r=0.86) and the AO group (r=0.76), and low, but significant, correlations for Cp and Zn in the AO group (r=0.66). The increase in the oxidase activity of Cp and concentration of Cu in serum in ischaemia is caused by the acute phase response. The relationship of Zn concentration and Cp activity in ischaemia is indirect and needs further study. Keywords: Chronic arterial occlusion, ischaemia, ceruloplasmin, Cu, Zn, serum.

Introduction Progressive narrowing of the arterial lumen due to the accumulation of lipids and fibrous tissue within the intimal layer of the aortic wall diminishes the circulation to Reprint requests to Dr. Maria Iskra, Katedra i Zaklad Chemii Og61nej, Akademia Medyczna im. Karola Marcinkowskiego, ul. Grunwaldzka 6, 60-780 Poznan, Polska

the lower extremities. A prolonged low level of perfusion may lead to chronic ischaemia and tissue necrosis. The presence of ischaemic tissue excites an inflammatory reaction which is mediated by interleukin 11-6 type cytokins, produced by monocytes, macrophages, fibroblasts, platelets and endothelium cells (1). The effects of inflammatory reaction (2,3,4), surgery (5) and the progression of atherosclerosis (6) on copper

Ceruloplasmin, Cu and Zn in chronic arterial occlusion of the lower limbs

enzyme activities have been widely discussed. Ceruloplasmin (Cp), the main protein binding copper in plasma, is one of the positive acute phase proteins (APhPs), whose concentration and activity in plasma increase following infection, inflammation or trauma (7,8). The main physiological functions of Cp are related to copper transport, oxidation of biogenic amines, oxidation of Fe 2+ to Fe3+ for uptake by apotransferrin and apoferritin, and antioxidant activity protecting lipids against free radicalinduced injury (9). The relationship between atherosclerosis and changes in APhPs in serum has been studied after cardiac surgery (5), coronary angiography (6), and in acute (10) and chronic arterial occlusion (11). The levels of C-reactive protein (CRP), (Xl-acid glycoprotein (AGP) and haptoglobin (Hp) were found to be the best markers among APhPs for the identification of inflammation, monitoring of the postoperative period and the progression of coro-

77

and rest pain (13 patients) and III - ankle pressure < 50 mm Hg and ulcer or necrosis of the lower limbs. Ischaemia in the group I was classified as moderate and both in group II and III as critical. The clinical characteristics of the patients are shown in Table 1. The control was a group of 25 blood donors, healthy males aged 22-49, who had undergone a medical checkup before the study. Blood was taken from all patients before surgery and in control group in the fasting state in the morning. The sera obtained were analyzed for concentrations of lipids and uric acid, as well as for the activities of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and ceruloplasmin; the remaining coded serum samples were stored at -20°C until the assay for concentrations of elements. The study was approved by the Ethics Committee of the University of Medical Sciences in Poznan, Poland.

nary atherosclerosis. The decrease in the plasma concentrations of negative APhP (albumin, transferrin, (X2-HS glycoprotein) results in a rise of free ligands such as fatty acids, hormones, vitamins and trace elements (8). Previous work had demonstrated higher concentrations of copper and zinc in the serum of men with atherosclerosis obliterans (AO) in comparison with control subjects and in the arterial wall in AO in comparison with abdominal aortic aneurysm (AA) (12). In the present study the severity of ischaemia of lower limbs has been studied in relation to the activity of Cp and concentrations of copper and zinc in the serum of men withAO.

Analytical methods

Total- HDL- and LDL-cholesterol (TC, HDL-C and LDL-C, respectively) and triacylglycerol (TAG) concentrations in serum were estimated by using the enzymatic kits from Boehringer Mannheim Biochemica. Concentra-

Table 1. Clinical characteristics of patients with chronic arterial occlusion (AO), according to the degree of ischaemia of the lower limbs Degree of ischaemia

n=8

II n=13

III n=l1

Age (a)

55.1 ± 8.1

58.2 ± 7.0

57.4 ± 9.7

Material and Methods

TC (mmol/l)

5.18±1.01

6.14± 1.26

5.65 ± 1.48

Patients

LDL-C (mmol/l)

3.44 ± 0.84

3.93 ± 1.14

3.80 ± 1.49

HDL-C (mmolll)

1.06 ±0.25

1.08 ±0.36

1.08 ± 0.32

TAG (mmol!l)

1.49 ± 0.59

2.34 ± 1.60

1.54 ± 1.18

ASAT (Ull)

25.6 ± 10.2

31.1 ± 18.2

26.5 ± 15.6

ALAT(U/I)

17.8 ± 9.7

22.0 ± 11.5

20.9 ± 13.3

Uric acid (gil)

39.4 ± 14.8

45.4 ± 12.6

56.7 ± 17.6*

Ca (mmol/l)

1.77 ± 0.20

1.86 ± 0.67

2.1O± 0.43

Mg(mmo/I)

0.76 ± 0.20

0.67 ± 0.14

0.62 ±0.13

The subjects of this study were 32 patients, males aged 43-73 years, with chronic arterial occlusion of the lower limbs (AO) who were admitted to the Department of General and Vascular Surgery at the University of Medical Sciences in Poznan, Poland, between July and December 1997. All patients underwent arteriography of the lower limbs and ultrasound measurement of ankle pressure, and were treated surgically (endarterectomies, vascular reconstruction). They were divided into three groups according to the degree of ischaemia of the lower limbs: I - ankle pressure ~ 50 mm Hg and intermittent claudication (8 patients), II - ankle pressure < 50 mm Hg

n=number of patients, the results are expressed as mean±standard deviation * significant difference between groups I and III, P<0.05

M. Iskra and W. Majewski

78

Table 2. Oxidase activity of ceruloplasmin (Cp) and concentrations of copper and zinc in the serum of men with chronic arterial occlusion (AO) with different degrees of ischaemia of the lower limbs and in a control group Oxidase activity Cu (!-lmolll) Degree of ischaemia of ceruloplasmin (U/l) Controls n=25

123.7

± 28.6

16.3

±

1.8

35

:s30 0

.§, 25 c

Zn (!-lmol/l)

,Q

~

15.2

..,.-\

.. " -,.

20

15 E (J)

± 2.3

•

u

c 10

0

18.5

± 3.3

14.5

± 4.3

± 84.2

22.2

± 3.6

19.1

± 7.1

± 3l.l

25.3

± 3.1

23,0

± 6.4

109.3

± 31.6

194.8

199.7

n=8

II n=13

*,**

III n=ll

*,**

**

*,** *.**

*,**

* significant difference in comparison with degree I of ischaemia, P<0,05 **significant difference in comparison with control group, P<0.05

U ::J

5

0

OL--+---------+--~--r_-+--~

o

50

100 150 200 250 300 350 400 Oxidase activity of Cp (UII)

Figure 2, Oxidase activity of ceruloplasmin (Cp) vs concentration of Cu in serum of men with chronic arterial occlusion (AO). Number of subjects = 32; correlation coefficient r=0.76, P<0.05.

Statistic calculations

tion of uric acid in serum was determined by the reaction with uricase by using diagnostic kit from Sigma Chemical Company. ASAT and ALAT activities were estimated by using the diagnostic kits from Boehringer Mannheim Biochemica. The oxidase activity of Cp in serum was determined according to the method of Schosinsky (13) by

Statistical calculations were carried out using Instat Sigma software. The means ± standard deviations were calculated for the results and the differences between groups were evaluated by Student's t-test or CochranCox's test. Pearson's correlation coefficients were calculated for the activity of Cp and concentrations of copper

using o-dianisidine as a substrate. Concentrations of copper, zinc, calcium and magnesium in serum were determined by means of flame atomic

and zinc in the groups studied. The statistical significance was accepted for P
absorption spectrometry using standard solutions of elements (l mg/ml of metal in I wt. % HCI or HN03) from Aldrich (14).

Results

The concentrations ofTC, LDL-C, HDL-C, TAG, Ca and Mg, and activities of ASAT and ALAT in sera of men

40

25

sa) 0

E 2, c 15 0

..-

'iii...

~ 10

~

....

• •

·f.-'"

.,:;

•

-

15

. -.

~

c

(J)

u

c 0

0

0

25

•

~ 20

c

::J

c 0

u u

35

'0 E 30

u

5

C

N

• . ... -•

. .•

•

..

10 5

0

0 0

25

00 75 100 125 100 Oxidate activity of Cp (UII)

175

200

Figure 1. The oxidase activity of ceruloplasmin (Cp) vs concentration of Cu in serum in the control group. Number of subjects = 25; correlation coefficient r=0.86, P<0.05

0

50

100

150

200

250

300

350 400

Oxidase activity of Cp (UII) Figure 3. Oxidase activity of ceruloplasmin (Cp) vs concentration of Zn in serum of men with chronic arterial occlusion (AO): Number of subjects= 32; correlation coefficient r=0.66, P<0.05

Ceruloplasmin, Cu and Zn in chronic arterial occlusion of the lower limbs

79

with different degrees of ischaemia of the lower limbs did

and necrosis includes the acute phase reaction induced by

not differ among the groups, as shown in Table 1. The

proinflammatory cytokines and, thus, the synthesis of Cp

mean concentration of uric acid was found significantly

and other APhPs. The proteic profile, including their as-

increased in the serum of men with the highest degree of ischaemia (III). The effect of the ischaemia of lower limb on the oxidase activity of Cp and concentrations of Cu and Zn in serum was observed in men with AO. The mean value of the oxidase activity of Cp in serum in the whole group of men with AO of the lower limb (173.1 ± 69.2 UII) was higher in comparison with the control group and in men

say, is useful as a clinical marker during early inflammation (8). The levels of CRP and AGP in serum studied both in acute and chronic arterial occlusion of the lower limb were found to be increased with the severity of ischaemia (10,11) and have been recommended as clinically useful in the monitoring of patients during the postoperative period, especially those with a CRP level high-

with critical ischaemia (degrees II and III) in comparison

er than 50 mill which is predictive of complications. CRP, AGP and HP are proteins whose changes in concen-

with mention a moderate level of ischaemia (degree I) (Table 2). The mean concentrations of Cu and Zn in serum in the whole group with AO (22.2 ± 4.2 and 19.1± 6.9 Ilmolll, respectively) were found to be higher than in the control group and in men with critical ischaemia (degrees II and III) of the lower limbs in comparison with the moderate level (degree I) (Table 2). The differences in the activity

tration during acute phase response are rapid and more pronounced than changes in Cpo The biochemical functions of Cp are related to the presence of 6-7 Cu ions in one molecule and include the oxidation of Fe 2+ to Fe3+, binding the latter to apoferritin and eliminating free ferrous ions from plasma. The ferroxidase activity of Cp has an antioxidant character in plasma and consists in the inhibition of the Fenton reac-

of Cp and the concentrations of Cu and Zn between men with degree I of ischaemia and the control group were not

tion and scavenging of free radicals such as superoxide O2- and hydroxyl OHo. The antioxidant action of Cp has been derived from the nonspecific antioxidant effect observed with almost any protein involved in a cupric ion-

significant. The relationships between the oxidase activity of Cp and the concentration of Cu and Zn in serum were estimated as a linear correlation. Significant positive correlation coefficients were calculated for the activities of Cp and concentrations of Cu in the control group (r=0.86, P<0.05) (Figure 1) and the AO group (r=0.76, P<0.05) (Figure 2). Significant, but low, positive correlation coefficients were calculated for the concentration of Zn and the activity of Cp (r=0.66, P<0.05) (Figure 3) and for the concentration of Cu and Zn (r=0.57, P<0.05) in serum of men withAO.

0

mediated lipid peroxidation, and from its ferroxidase activity, both of which have been proposed as crucial functions during acute phase responses (16). Recently, the role of endogenous Cp as a source of transition metal ions involved in low density lipoprotein oxidation has been suggested (17,18,19) and a free metal ion-dependent mechanism was observed in vivo (15). Al-

The role of Cp and its oxidant activity in inflammation and other pathological processes, in human vascular disease especially, is not well known (15). The present study showed that in chronic ischaemia of the lower

though there is no evidence for the presence of Cp in the arterial wall, specific receptors for it have been described in aortic extracts (17) and ferroxidase I activity due to Cp has been determined in atherosclerotic lesions (20). Studies of the content of advanced human atherosclerotic lesions showed the presence of Cu and Fe ions, which can support the oxidation of LDL mediated by macrophages (21). The prooxidant site of human Cp has been localized and the crucial role of histidine (His) in position 426 in the second domain of Cp was indicated (15). The model depicting on exposed Cu ion the surface of Cp may also

limbs there is an increase in concentration of Cu and Zn and in the oxidase activity of Cpo The rise in serum Cu with the degree of ischaemia is secondary to the changes in Cp and caused mainly by the increase in the concentration of Cp (immunologically detected in the acute phase reaction (2)) and its activity, as in present study. The body defence system against infection, inflammation, trauma

explain the oxidation function of other Cu-containing proteins, in particular of Cu, Zn-SOD. The relationship of the concentration of zinc and the oxidase activity of Cp in serum was supported by a significant correlation coefficient (r=0.76), which was observed only in AO, but not in the control group. The reason for this is unknown. Cp does not bind Zn in serum,

Discussion

80

M. Iskra and W. Majewski

and the indirect relationship of increased Zn and the activity of Cp in the acute phase response in ischaemia possibly results from changes in concentrations of other proteins that bind Zn such as albumin, a 2-macroglobulin (a-MG), transferrin (Tf) and histidine-rich glycoprotein. Albumin and Tf bind approximately 60% and 12% of the Zn present in plasma but both proteins should decrease the Zn level in plasma as they are negative APhPs. The increased level of Zn in serum in critical ischaemia may be caused by changes in the a-MG concentration, a protein responsible for binding and transport of approximately 20% of the Zn present in plasma. Activated a-MG binds host and foreign peptides and particles, thereby serving as a humoral defense barrier against pathogens in the plasma and tissues (22). Changes in Zn metabolism and interleukin-l beta (IL-l) release were observed as a part of the physiological response to tissue injury and trauma and to low-dose 11-1 beta administration in rats (23). The increased level of Zn in plasma in ischaemia may also result from its role in the signaling pathway involved in apoptosis and its anti atherogenic properties which prevent derangements of the vascular endothelium (24). The role of Zn in ischaemia as cytoprotective by decreasing the formation of OR· radicals has also been suggested (25). The increased concentration of Cu in serum in ischaemia of the lower limb is caused by the acute phase response, which is confirmed by the increase in the oxidase activity of Cpo The reason for the parallel increase in Zn concentration cannot be explained by changes in the APhP levels and possibly this is due to a shift of free Zn ions from intracellular to extracellular fluid or to an increase in the level of Zn-dependent enzymes.

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