ARTICLE
Auteur(s) : M
Iskra1, D Barałkiewicz2, W
Majewski3, M Pioruńska-Stolzmann1
1Department of General Chemistry, Poznań University
of Medical Sciences, Poland
2Faculty of Chemistry, Adam Mickiewicz University,
Poznań, Poland
3Department of General and Vascular Surgery, Poznań
University of Medical Sciences, Poland
Chronic ischemia of the lower limbs results from a decreased supply
of oxygen and nutrients due to atherosclerosis obliterans. Ischemia
of a critical degree invokes an inflammatory response, activation
of immune cells, increased production of interleukin-6 (IL-6) and
acute phase proteins (APhPs) as a response to tissue destruction.
Surgery and complicated postoperative treatment may also result in
an increase in the concentration of cytokines and APhPs. Ischemia
affects the integrity of the cell membrane, the permeability of
blood microvessels, concentrations of intracellular and
extracellular electrolytes and free radical generation. Changes in
electrolyte concentrations in blood plasma and tissues have been
observed in cardiovascular diseases, atherosclerosis, arterial
hypertension and diabetes mellitus.Inflammatory changes, occurring
in patients with chronic ischemia and undergoing surgical
treatment, may alter macro- and trace element concentrations in
blood. Serum magnesium concentration was found decreased in some
patients with myocardial infarction [1], after heart surgery [2],
and traumatic injury [3]. Hypomagnesemia, with total plasma Mg
concentration below 0.75 mmol/L, was shown in one third of the
critically ill postoperative patients [4]. Dietary magnesium
deficiency associated with oxidative stress was recognized as a
pathogenic factor in inflammatory processes, cardiovascular
pathology, and ageing [5, 6]. Copper is essential for maintaining
the structure and function of some proteins and antioxidants, and
seems to be involved in the progress of atherosclerosis. Although
deficiency or marginal intake of Cu has been proposed as a risk
factor for cardiovascular diseases, serum Cu concentration was
found to be increased in atherosclerosis obliterans (AO) [7].
Severe loss of serum Zn and a significant increase in Cu were
observed during inflammation [8-10] and after cardiopulmonary
bypass [11]. Zinc involvement in the inflammatory processes may
result from its indispensability for the activity of Cu,Zn-SOD
[12], the induction of metallothionein synthesis, and the
stabilization of cell membrane [13]. The mechanism of Zn
antioxidant properties remains unclear, but the stimulation of
reactive oxygen species (ROS) generation was observed in Zn
deprivation [14].The aim of the present study was to investigate
the modification of Mg, Cu and Zn concentrations in serum of
inpatients with AO before surgery and during the postoperative
treatment, and the effect of chronic ischemia of lower limbs on the
relationship between elements.
Materials and methods
Subjects
The studied group consisted of 54 men with chronic ischemia of the
lower limbs due to AO. Patients were admitted to the Department of
General and Vascular Surgery at University of Medical Sciences in
Poznań, Poland. The age of patients ranged from 40 to 83 years
(58.5 ± 11.0). In all patients aortography and ultrasound
measurement of ankle blood pressure was performed before surgery
(vascular reconstruction). Patients were divided into two subgroups
according to the degree of ischemia of lower limbs: moderate
ischemia (MI) – intermittent claudication and ankle systolic blood
pressure above 50 mmHg (25 patients), and critical ischemia
(CI) – rest pain and/or ulceration or necrosis, and ankle systolic
pressure below 50 mmHg (29 patients). The classification was
done according to the ankle systolic pressure index following the
European Report of Critical Limb Ischemia [15, 16]. Patients were
not supplemented with Mg, Cu and Zn before or after surgery, and
those with hypertension, cancer and kidney diseases were excluded
from the study. The control group comprised 24 healthy male blood
donors, aged 20-59. They underwent a routine medical checkup before
the blood collection was done.
Blood samples were collected in the fasting state from the
brachial vein in the group of men with AO and in the control group.
In AO group, blood samples were collected before surgery and in the
postoperative period of 1-4, 7-12 and after 12 days of treatment.
The group of patients with AO studied during the postoperative
treatment was 50, i.e. 4 patients less than before surgery. In
order to analyse the results obtained during the postoperative
treatment for a more consistent group of patients, 4 patients (one
with MI and three with CI) were excluded by reason of complicated
postoperative treatment (second surgery, myocardial infarction,
cancer). The study was approved by the Ethical Committee at the
University of Medical Sciences in Poznań, Poland.
Methods
The concentration of Mg, Cu and Zn in serum was determined by using
Varian SpectrAA Plus atomic absorption spectrometer with deuterium
background correction and a GTA-96 graphite furnace. Certified
reference material (Trace elements in serum, level 1) from LGC
Promochem, UK, was used.
Concentration of interleukin-6 (IL-6) was measured by ELISA
(Quantikinine, R&D Systems, Minneapolis, USA), and those of
C-reactive protein (CRP), α1-glycoprotein (AGP) and
ceruloplasmin (Cp) with rocket immunoelectrophoresis by using
rabbit antibodies (Dakopats, Copenhagen, Denmark) and human
standard serum (Behrinhwere AG Marburg, Germany).
Oxidase activity of Cp in serum was measured
spectrophotometrically after incubation with o-dianiside as a
substrate according to Schosinsky [17].
Element and Cp concentrations and the activity of Cp (expressed
as mean ± standard deviation) were compared by Student’s t-test.
The results for IL-6, CRP and AGP (presented as medians) were
compared between groups using Mann-Whitney unpaired test.
Results
The mean values from baseline (0 days) determinations of Mg, Cu and
Zn, namely before surgery, for the whole AO group, MI group
separately from CI group, and for the control group are presented
in table 1( Table 1 ). The mean
values of serum Mg and Cu concentrations in men with AO were found
significantly different in comparison with the control group.
Before surgery, increased Cu concentration and decreased Mg were
determined, followed by a subsequently lower concentration ratio
Mg/Cu and higher Cu/Zn in AO than in the control group. Ischemia of
the lower limbs affects the concentration of Cu, but not that of
either Mg or Zn. In men with critical ischemia the mean values of
serum Cu concentration and the ratio Cu/Zn were found higher, when
compared with the group of moderate ischemia.
Levels of inflammation markers and APhPs respond to a chronic
state of ischemia of the lower limbs. IL-6, CRP and AGP
concentrations (expressed as medians) before surgery were observed
to be higher in CI than in MI patients. The mean Cp concentration
and its oxidase activity were found significantly increased in CI
as compared with MI (table 2( Table
2 )).
During the postoperative treatment a significant increase in the
mean serum Cu concentration after 12 days (C3), and in
the mean Zn concentration after the period of 7-12 days
(C2), were observed (table 3( Table 3 )) when compared with the values found
before surgery (C0).
The changes in Mg, Cu and Zn concentrations in the postoperative
treatment were calculated for three periods of 1-4
((Δ1), 7-12 (Δ2) and more than 12 days
(Δ3) after surgery according to the formula:
Δx = Cx – C0. Respective
concentration changes (Δx) for each element were
calculated. For the changes in Cu (Δ1, Δ2 and
Δ3) and Zn concentrations (Δ2 and
Δ3) the negative correlation coefficients rx
(C0 vs Δx) with the concentration of the
element before surgery (C0) were calculated
(table 3).
Table 1 Mg, Cu and Zn concentrations and their ratios
in serum of men with atherosclerosis obliterans (AO), with critical
(CI) and moderate ischemia (MI) of lower limbs due to AO, and in
control group.
|
Group
|
Mg (mmol/L)
|
Cu (μmol/L)
|
Zn (μmol/L)
|
Mg/Cu
|
Mg/Zn
|
Cu/Zn
|
|
|
|
14.0 ± 4.5
|
|
59.4 ± 20.4
|
|
|
0.84 ± 0.26
|
|
13.8 ± 4.4
|
|
|
|
|
|
|
14.2 ± 4.5
|
|
56.1 ± 21.1
|
1.28 ± 0.42
|
|
0.89 ± 0.13
|
16.1 ± 2.2
|
14.6 ± 2.1
|
53.6 ± 12.8
|
52.7 ± 14.0
|
1.18 ± 0.30
|
Table 2 Concentration of IL-6, CRP, AGP (median), Cp
and the oxidase activity of Cp (mean ± SD) in serum of men with
lower limb critical (CI) and moderate ischemia (MI).
|
Group
|
IL-6
|
CRP
|
AGP
|
Cp
|
Cp
|
|
ng/L
|
mg/L
|
mg/L
|
mg/L
|
U/L
|
|
MI
|
24
|
1
|
720
|
442 ± 186
|
123.1 ± 38.3
|
|
n = 25
|
|
CI
|
62*
|
44*
|
1045*
|
655 ± 175*
|
177.8 ± 63.1*
|
|
n = 29
|
Table 3 Concentrations of Mg, Cu and Zn in serum of men
with AO before surgery and in the postoperative treatment (n =
50).
|
Element
|
Days after surgery
|
Cx (mean ± SD)
|
Δx
|
r (C0 vs Δx)
|
|
Mg
|
0
|
C0
|
0.80 ± 0.20
|
|
|
|
|
1 – 4
|
C1
|
0.83 ± 0.25
|
Δ1
|
-0.030
|
r1 = -0.2407
|
|
mmol/L
|
07-12
|
C2
|
0.87 ± 0.30
|
Δ2
|
0.003
|
r2 = -0.2670
|
|
> 12
|
C3
|
0.83 ± 0.29
|
Δ3
|
0.049
|
r3 = 0.3197
|
|
Cu
|
0
|
C0
|
21.7 ± 3.8
|
|
|
|
|
1 – 4
|
C1
|
21.3 ± 6.2
|
Δ1
|
-0.451
|
r1 = -0.5768 **
|
|
μmol/L
|
07-12
|
C2
|
22.6 ± 5.8
|
Δ2
|
0.928
|
r2 = -0.6759 **
|
|
> 12
|
C3
|
25.1 ± 5.0 *
|
Δ3
|
5.238
|
r3 = -0.7948 **
|
|
Zn
|
0
|
C0
|
13.8 ± 2.9
|
|
|
|
|
1 – 4
|
C1
|
13.8 ± 4.3
|
Δ1
|
0.050
|
r1 = -0.1477
|
|
μmol/L
|
07-12
|
C2
|
16.6 ± 3.7 *
|
Δ2
|
1.466
|
r2 = -0.5324 **
|
|
> 12
|
C3
|
13.9 ± 2.3
|
Δ3
|
0.671
|
r3 = -0.8223 **
|
Discussion
Increased levels of chosen biomarkers of acute phase reaction,
found in patients with CI as compared with the MI group
(table 2), indicate inflammation while experiencing severe
ischemic changes in the lower limbs. Ischemia-reperfusion events
and inflammatory responses cause increased production of reactive
oxygen species and alterations of macro- and trace element
concentrations [18]. The impact of chronic lower limb ischemia and
the postoperative treatment on Mg, Cu and Zn in human serum has not
been considered efficiently. The modulatory effect of Mg status on
immune cell function has been observed in vitro [19] and on
reperfusion injury in patients with acute myocardial infarction
pretreated with Mg sulfate [20]. The nutritional status plays an
important role in modification of diseases related to inflammation
[21]. An early consequence of Mg deficiency recently observed in
Mg-deficient rats was a significant increase in IL-6 in plasma,
suggesting that reduced extracellular Mg might be responsible for
the activated state of immune cells [22]. Serum Mg concentration in
humans is often observed normal despite depletion of its total
level in the human organism [23]. Lower Mg found in serum of the
group of patients studied with AO, in comparison with the control
subjects, may influence numerous regulatory functions of
Mg2+ ions in hormonal, cardiovascular and immune
systems. Depletion of Mg in plasma induces higher susceptibility of
the lipoproteins to the oxidative stress, and a possible
pro-oxidant effect [24, 25]. Mg plays a critical role in the
function of mitochondrial ATP and its decrease in ischemia may
suggest inadequate ATP synthesis, and lower level of ATP for the
metabolic processes. There are contradictory observations of the
impact of ischemia on serum Mg concentration [10, 26]. In the
present study Mg concentration in serum was not significantly
influenced by ischemia and was maintained at a similar level during
the postoperative treatment. It may only be concluded that the
measurement of intracellular Mg concentration in the erythrocytes
and the estimation of the ratio of ionized/total Mg plasma
concentrations in patients with ischemia would better reflect Mg
changes in the inflammatory response.
Ionized Mg (i-Mg) should be considered as more relevant clinical
marker of Mg metabolism, and i-Mg in serum has been suggested by
other authors to reflect more adequately the status of Mg in blood.
However, the insufficient quality and the lack of standardized
procedure for i-Mg measurement with the selective electrodes means
that i-Mg is currently not used in clinical practice [27].
Moreover, there is strong evidence that total-Mg (t-Mg) and i-Mg
are very closely related. Therefore, serum t-Mg is the most
practical and commonly used parameter for assessing disorders of Mg
metabolism in clinical practice [28]. In the present study, the
t-Mg in serum was determined because we intended to follow the
total Mg changes in the postoperative treatment, and find possible
relationships with levels of other elements (Cu, Zn). Dramatic
changes in Mg after surgery, and differences between the AO and the
control groups were not observed. Even under such deleterious
conditions as ischemia of different degrees may cause, no
statistically significant difference was found in the total Mg
concentration in serum between MI and CI groups.
It may only be suggested that t-Mg in serum represents quite a
stable value, regulated homeostatically, and its level should not
be recognized as a marker of atherosclerotic and ischemic
alterations to the whole organism. On the other side, the
elucidation of the mechanism of Mg homeostasis in atherosclerosis
and ischemia needs further study, including the ionized and total
Mg determinations. It is suggested that a speciation of Mg in blood
should be performed, i.e. find and determine free and bound forms
of Mg(II) ions and follow their changes during the postoperative
treatment.
Dietary deficiency or marginal intake of Cu was recognized as a
risk factor for cardiovascular diseases [29]. However, in serum of
patients with critical ischemia of the lower limbs due to
atherosclerosis, the Cu concentration was found to be significantly
increased and positively correlated with the oxidase activity of
Cp, not only in this study, but also in all previously studied
groups of patients with critical ischemia of the lower limbs due to
AO [30-32]. The increase in serum Cu and the activity of Cp, an
APhP in plasma, was also found in patients with chronic lower limb
ischemia [33] and in patients with coronary heart disease
undergoing coronary angioplasty [34]. The present results show that
the postoperative treatment may affect serum Cu concentration, and
also, what is more interesting, the magnitude of changes is in
inverse relation to the level before surgery.
Decreased plasma Zn concentration was observed in the
inflammatory state and seemed to be a result of the modification to
Zn distribution in the organism and a protective mechanism in
inflammation [35]. The immune dysfunction in Zn deficiency involves
an increase in T lymphocytes and cytokine production followed by
enhanced free radical generation. Zinc may be capable to retard
oxidative processes through different mechanisms, namely as a
component of the antioxidants: Cu,Zn-SOD and metallothioneins, and
an antagonist of copper [14]. A deficiency of Zn in rats stimulates
the generation of reactive oxygen species by decreased Cu,Zn-SOD
activity and results in oxidative damage [19]. The results of the
present study showed that AO and ischemia did not affect Zn levels
in serum. A rise in Zn observed at 7-12 days after surgery followed
by normalization may suggest a shift of Zn from tissues or
erythrocytes to blood plasma as a consequence of an increased
demand for this element after surgery. Moreover, Zn concentration
seems to be homeostatically regulated as the changes in Zn observed
during treatment were found inversely correlated with the
concentration of Zn before surgery. Speciation of Zn in
plasma/blood during the postoperative treatment might help to
elucidate this phenomenon. The level of Zn in erythrocytes and
concentration of α2-macroglobulin, another acute phase
reactant, should be also analyzed.
It was shown previously that the ratio Mg/Cu is affected by
ageing, atherosclerosis, and the degree of ischemia, and its value
differs between serum and the arterial wall [26]. It was also
suggested that serum Mg/Cu might be a marker of distorted
equilibrium between Mg and Cu, because it reflects their
relationship better than element concentrations. In this study it
was shown that the value of the serum Cu/Zn ratio also differs
between control and atherosclerotic subjects, and increases in
critical ischemia when compared to moderate degree ischemia. It may
be postulated that the ratios Mg/Cu and Cu/Zn may be markers of the
impaired relationships between those elements in atherosclerosis
and ischemia.
The present results showed the inverse relationship between
baseline concentration both Cu and Zn in serum and their changes in
the postoperative treatment. It may give rise to further questions
about the effect of the magnitude of inflammation on the regulation
of Cu and Zn concentrations, and also other trace elements, in
circulation in patients with lower limb ischemia.
The correlations found for both Cu and Zn before surgery and
changes observed during the postoperative treatment may suggest a
modulatory mechanism for Cu and Zn serum concentrations after
surgery, i.e.the lower value before surgery may result in a rise in
Cu and Zn concentrations after surgery (and vice versa).
The importance of some nutrients for the immune system and the
antioxidant status of the human body support the opinion that the
sequence of events leading to the modification of macro- and trace
element concentrations during ischemia and the inflammatory
response needs to be elucidated. The relationship between Mg, Cu
and Zn concentrations in monitoring the postoperative treatment
needs more consideration.
Conclusions
Chronic ischemia of the lower limbs affects serum concentrations of
Mg and Cu, and the ratios Mg/Cu and Cu/Zn. Critical ischemia of
lower limbs increases serum Cu concentration, and the oxidase
activity of Cp, the main protein carrier of Cu in plasma. It may be
postulated that the ratios Mg/Cu and Cu/Zn may be markers of the
impaired relationships between those elements in atherosclerosis
and ischemia. Postoperative treatment results in changes in Cu and
Zn concentrations that are inversely related to the levels before
surgery.
Acknowledgements
This work was supported by a Grant No 502-2-90-07 from
Poznań University of Medical Sciences and Adam Mickiewicz
University in Poznań, Poland.
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