ARTICLE
Auteur(s) : A
Korycińska1, W Dąbrowski2, Z
Rzecki2, M Dragan1, P Pożarowski1,
J Wrońska3, J Stążka4, K
Pasternak3, J Roliński1
1Department of Clinical Immunology Medical
2Department and I Clinic of Anaesthesiology and
Intensive Care
3Department of General Chemistry
4Department of Cardiosurgery Feliks Skubiszewski Medical
University of Lublin, Poland
It is well known, that magnesium (Mg) is a very important
intracellular electrolyte for cell homeostasis [1]. Mg acts as a
cofactor of many enzymes and is involved in a variety of biological
functions, including structural and regulatory intracellular
balance. Mg deficiency is connected with the development of heart
diseases including hypertension and atherosclerosis. This
electrolyte stabilizes mitochondrial linkage and regulates
oxidative and ATP synthesis processes [2, 3]. Hypomagnesemia may
cause changes in mitochondrial transmembrane potential and
deregulates the mitochondrial electrolyte balance [4]. It may lead
to accumulation of sodium and calcium ions in intracellular spaces
which may be particularly important for the disturbance of
mitochondrial respiratory activity (regulated by calcium ions) [1,
5]. These changes cause hyperproduction of reactive oxygen species
(ROS) [6, 7] and many authors suggest that the ROS accumulation
results in programmed cell death or apoptosis [8]. It has also been
reported that an increase in intracellular calcium and magnesium
concentration promotes apoptosis [9].Apoptosis is an active and
physiological mechanism allowing cells to die in a controlled and
organized manner with characteristic biochemical and
ultrastructural changes. Apoptosis of immunocompetent cells such as
lymphocytes is involved in the regulation of the immune response,
particularly under stress conditions. Extensive trauma, surgical
stress and general anaesthesia cause neuroendocrine and
inflammatory activation with hyperproduction of catecholamines, as
well as proinflammatory cytokines such as IL-6, TNF-α and this may
affect the immune system. Apoptosis of lymphocytes plays an
important role in the resolution of these processes and is partly
secondary to their activation and with reference to T lymphocytes,
is described as activation-induced cell death (AICD) [10]. There
are many morphological and biochemical hallmarks of apoptosis that
can be detected and measured with cytometry and other techniques.
Such cells may be characterized by the collapse of the
mitochondrial transmembrane potential (ΔΨm), which is
considered to be the first detectable sign of apoptosis.
Mitochondria are organelles with two well-defined compartments: the
matrix, surrounded by the inner membrane and the intermembrane
space, surrounded by the outer membrane. The inner membrane
contains various molecules such as ATP synthases, adenine
nucleotide translocators and electron transport chains, each of
them being dependent on magnesium. Permeabilization of inner
membranes leads to changes in mitochondrial membrane potentials
[10]. It has been found that alterations in mitochondrial function
play a key role in the effecter phase of apoptosis induced by
different agents [2, 10, 11]. These alterations include the
disruption of ΔΨm, the generation of ROS and the opening
of permeability transition pores (PTP) in the outer mitochondrial
membrane through which pro-apoptotic factors such as cytochrome c
are released into the cytoplasm. ΔΨm can be easily
measured by the use of some cationic lipophilic dyes such as
chloromethyl-X-Rosamine (CMXRos) [12].Many authors underlined that
surgery and anaesthesia cause depression of cell - mediated
immunity in the postoperative period [6, 13]. Apoptosis of
lymphocytes and the resulting susceptibility to infections from
common pathogens seem to be a particularly important problem during
and after cardiac surgery with extracorporeal circulation (ECC).
Probably a multistage character of ECC procedures as well as
intraoperative therapy and electrolyte disturbances may contribute
to this process.The aim of this study was to assess the correlation
between apoptosis of lymphocytes and Mg blood concentrations during
CABG procedure and in the early postoperative period.
Material and methods
The study was approved by the Bioethical Committee of the Feliks
Skubiszewski Medical University of Lublin; Poland (No
KE-0254/28/2003) and included the patients treated for stable
angina pectoris (I° and II° according to Canadian Cardiovascular
Society, or CCS) and scheduled for elective aortocoronary bypass
(CABG-coronary artery bypass graft) with ECC.
In the evening preceding the operation the patients were
premedicated with lorazepam and promethazine. One hour before
anaesthesia all the patients received lorazepam and morphine. The
patients underwent general anaesthesia with fentanyl, midazolam,
and etomidat. Muscle relaxation was obtained by injecting a single
dose of pancuronium. The anaesthesia was maintained throughout the
procedure using midazolam-fentanyl infusion and inhalatory
fractionated doses of Izofluran. During the implantation of
aortocoronary by-passes, circulation and ventilation were
maintained by a heart-lung machine SIII (Stockert). The following
substances were used for priming: Ringer solution, HAES solution,
20 % mannitol, Natrium bicarbonatum 20 mL and heparin
75 mg. The same composition of priming was used in all
patients. Cardioplegia was prepared using 0.9 % salt solution
supplemented with 3 g of potassium chloride and 20 mL of
sodium hydroxycarbonate. The degree of normovolemic hemodilution
induced by a constant volume of priming (1 800 mL) was
determined on the basis of haematocrit measurements and body
weight. During surgery the patients received supplementation of
potassium chloride to the level of 4.94 mmol/L ± 0.5.
All the patients were transported to the Postoperative Intensive
Care Unit immediately after the procedure, where they received a
short-term infusion of 5% glucose solution with insulin and 3 or
6 g of potassium chloride. None of the patients received Mg
infusion during surgery or the postoperative period.
Peripheral blood samples were taken at seven different stages:
1) just before anaesthesia, 2) 2 hours after the beginning of
surgery, 3) immediately after surgery, 4) 12 hours after the
beginning of surgery, 5) 24 hours after the beginning of surgery,
6) 36 hours after the beginning of surgery, 7) 54 hours after the
beginning of surgery. The samples were immediately transferred to
the laboratory for detection of apoptotic cells.
Mononuclear cells were isolated by density gradient
centrifugation (Gradisol, Aqua-Medica, Poland). Peripheral blood
serum samples were taken and stored at -20oC until
use.
The dissipation of ΔΨm was measured by the use of
CMXRos (Mito Tracker Red CMXRos, Molecular Probes). Cells were
incubated with the dye alone for 15 minutes at 37oC, for
the next 15 minutes monoclonal anti-glycophorin A FITC-conjugated
antibody (DAKO, Denmark) was added to contaminating lymphocytic
population. Just after the performance of these procedures, cells
were acquired and analyzed by flow cytometry (FACSCalibur, Becton
Dickinson, USA) with Cellquest Software by the same company.
The blood magnesium concentrations were determined by
spectrophotometric methods.
The Wilcoxon paired test was used to analyse differences between
the percentages of apoptotic cells and Mg concentrations, in
comparison with stage 1. The Spearman rank correlation was
used for the estimation of correlation between percentages of
apoptotic cells and Mg concentrations. The p-value < 0.05 was
considered significant. Data were presented as median and ranges.
Statistica 6 software was used for all statistical
calculations.
Results
The examinations were conducted in 20 men aged 53-70 (61.1 ± 6.9).
Sixteen patients had myocardial infarction during the past 3 years
and 18 were treated due to concomitant arterial hypertension (I° or
II° according to WHO classification). None of the patients was
treated for endocrinological, neurological and other systemic
diseases nor was resuscitated because of circulatory arrest.
The mean duration of the procedure was 205 min ± 35 and of
anaesthesia 235 min ± 30. In all the patients the aorta was
typically clamped and the mean closure time was 45.1 min ±
15.5. The aorto-coronary anastomosis was performed in shallow
hypothermia at 34.5°C ± 0.4. In all the cases the heart-lung
machine disconnection was uneventful and there was no need of
intra-aortic contrapulsation. Four patients did not require
pharmacological support after the end of ECC, seven were subjected
to continuous dopamine infusions in doses adjusted to their
clinical condition and nine had dobutamine infusion.
The significant increase of lymphocyte apoptosis (LA) was noted
from stage 2 to stage 7. A small decrease of LA degree was noted in
stage 5, but the value was significantly higher in comparison with
stage 1.
The blood magnesium concentrations significantly decreased in
stages 2 and 3. The level of magnesium decreased in stage 5 but
this change did not reach statistical significance (( figure 1 )).
There were significant negative correlations between Mg blood
concentration in stages 2 and 3 and late apoptosis of lymphocytes
in the stage 5 (lymphocyte apoptosis occurs dependently after Mg
decrease) (( figure 2 )).
Discussion
The concentration of magnesium in the cells depends on levels of
binding substances including nucleid acids, ATP or phospholipids,
although the main factor regulating intracellular concentration of
Mg is its concentration in serum. On the other hand the changes in
serum Mg concentrations during ECC are not explicitly defined and
recent reports stress the importance of the maintenance of
normomagnesemia, particularly in patients with stunned myocardium
[14, 15]. Our studies showed that patients undergoing surgical
myocardial revascularization with ECC have a high risk of decreased
serum magnesium concentrations. Polderman and Girbes [16] suggest
that the mechanism responsible for this may be a combination of
increased urinary excretion and intracellular shift, induced by a
multistage character of ECC procedures. A decrease in body
temperature during surgery and high urinary magnesium excretion
play the main roles in this pathology. Probably a kidney tubular
dysfunction results in urinary magnesium excretion [16].
Furthermore, hypomagnesemia during CABG is attributed to degree of
hemodilution [17]. Examining the changes in blood magnesium
concentrations in cardiosurgical patients, Satur et al. [17]
observed that initiation of normovolemic hemodilution caused a
17.3% decrease in serum Mg levels, which persisted until the first
postoperative day. They concluded that the main reasons for
magnesium depletion are: the most important – normovolemic
hemodilution and secondly – intraoperative and postoperative
cellular depletions.
Magnesium deficiency in serum results in relatively low
intracellular magnesemia leading to cell dysfunction. Through its
regulatory effects on sodium-potassium pump and ATPase, Mg directly
affects intracellular concentration of potassium and calcium and
its increased extracellular levels favourably influences the cell
tolerance to ischaemia and reperfusion. It is worth stressing that
decreased Mg level in blood leads to increased permeability of cell
membranes and reduces ΔΨm. This effect seems to be
relevant in cases of programmed cell death [18, 19]. Examining the
pathophysiology of changes in ΔΨm in rats, Marcocci et
al. [18] observed a significant decrease in intracellular Mg
concentration and simultaneous calcium ions (Ca) accumulation
leading to cell oedema. According to Lang et al. [19] this
mechanism is responsible for the initiation of cell apoptosis. A
decrease in intracellular magnesium concentration results in a
dysfunction of the sodium-potassium as well as magnesium-calcium
pump [4, 20] leading to intracellular calcium accumulation and cell
oedema [3, 21]. This leads to metabolism disorders and ROS
accumulation, which also favours apoptosis [19, 21, 22]. On the
other hand Chien et al. [4] suggest that achieving high levels of
free intracellular magnesium is central to the apoptotic process.
In their opinion magnesium mobilisation is an early event in cell
suicides and disruption of ΔΨm is dependent on this
phenomenon. However the release of magnesium from the mitochondria
could merely be a consequence of the opening of mitochondrial pores
as ΔΨm is reduced. Bossy-Wetzel et al. [23] imply that
this magnesium release is independent of the loss of
ΔΨm. According to them the release of mitochondrial
cytochrome c is reported to occur prior to, and to be independent
on, the disruption of ΔΨm, on the other hand the
cytochrome c release is strictly dependent on magnesium ions. Thus
it may be supposed that the acute disorders of magnesium play an
important role for the apoptotic process. However, it is difficult
to determine explicitly the precise cause of this cell pathology
during CABG. According to Maeno et al. [21] and Bortner and
Cidlowski [22], the other main reason for such changes is the
initial normotonic shrinkage of the cell caused by hyperosmotic
stress. Considering the above, it can be supposed that normovolemic
hemodilution used intraoperatively disturbs lymphocyte homeostasis
stimulating apoptosis. The examinations performed seem to confirm
this hypothesis as significantly increased apoptosis was already
observed during extracorporeal circulation procedures. Korycinska
et al. [24] demonstrate that the degree of lymphocyte apoptosis is
dependent on the degree of normovolemic hemodilution. They report
more marked cell suicides in patients with a high dilution of
blood. On the other hand, it may seem that the process in question
was not only initiated by normovolemic hemodilution. Many authors
stress unfavourable, apoptosis-stimulating effects of commonly used
anaesthetics [13, 25, 26]. According to Delogu et al. [8, 25, 26],
both operative stress and the drugs used initiate lymphocyte
apoptosis. Therefore, it may be supposed that the fentanyl and
pancuronium used in our study intensify apoptosis. Examining the
effects of opiods on LA, the authors mentioned above observed that
the opioid already disturbed the lymphocyte ΔΨm in the
90th minute of cell exposure to fentanyl, and the
highest intensification was noted in 120th minute. It is
also noting that the intracellular changes observed were
accompanied by excessive ROS production, which may confirm the
intracellular pathology described earlier [8, 26]. However, it is
difficult to compare these results with our findings as all our
patients received continuous fentanyl infusions throughout the
operation and anaesthesia.
Analyzing LA intensification during extracorporeal circulation
procedures, one should not neglect the effects of the procedure
itself. One of the most harmful factors impairing cell homeostasis
are filters and oxygenerators. It may seem that the extent of
damage and cell apoptosis is likely to depend on the kind of the
oxygenerator used. However, Lears et al. [27] who studied the
effects of various oxygenerators on the extent of lymphocyte
apoptosis, did not find relevant differences in “lymphocyte
response” to the procedure with cardiopulmonary bypass. According
to them, LA intensification results from the procedure itself
rather than from the equipment used.
The double phase of LA is interesting although difficult to
explain. It may result from the simultaneous activation of
caspase-dependent and caspase-independent pathways by different
factors such as surgical stress or mechanical damage [28]. Possible
mechanisms for these phenomenon are still discussed, including:
cell – cell interactions, intracellular hydrodynamic disturbances
and waste accumulation [29, 30]. Postoperative treatment –
particularly dopamine or dobutamine infusion is not without
importance too [31]. Ciocia et al. [31] in their analysis of the
suicide of cells incubated in different dopamine or dobutamine
solutions, presented dose-dependent increases of LA, with the
highest intensity observed after 24 hours of incubation.
Interestingly, the pathology was inhibited by a β-blocker –
propranolol. Thus we believe that the second increase of LA
observed by us may have resulted from dopamine or dobutamine
infusion, although this correlation in patients after
extracorporeal circulation is not explicitly documented and
requires further studies.
The negative correlation between blood Mg level and LA intensity
is also difficult to explain. It seems that high Mg blood
concentrations may confirm the protective effects of Mg on the
cell, particularly in stress situations. Studying the effects of
high magnesium levels in blood on nerve cells, Park and Hyun [32]
noted significantly weaker nerve cell apoptosis in patients treated
with high doses of Mg. Likewise, Fernandez-Gomez et al. [33] stress
the beneficial effects of high magnesium levels on ΔΨm.
According to them, this element substantially inhibits
mitochondrial membrane damage and ROS production, thus decreasing
the extent of cell apoptosis. In our study the negative correlation
between Mg blood concentration in stages 2 and 3 and apoptosis of
lymphocytes in stage 5 confirm the opinion that hypomagnesemia may
lead to increased apoptosis even several hours later. Thus it seems
that LA observed in our study may result both from the operative
procedure itself and from changes in blood magnesium levels.
Conclusions
1. The extracorporeal procedure caused a decrease of magnesium
blood concentrations and an increase of lymphocyte apoptosis.
2. The decrease of magnesium concentration in the blood is one
of the causes of increased lymphocyte apoptosis.
3. The lymphocyte apoptosis has a two-phase course after
extracorporeal circulation.
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