Abstract
Magnesium is one of the most important factors for regulation of inflammatory response as well as muscle function, and COPD is a multicomponent disease characterized by abnormal inflammatory response of the lungs with systemic muscle dysfunction. Because polymorphonuclear (PMN) cells are significantly represented in the pathogenesis of COPD, concentrations of total (tMg) and ionised magnesium (iMg) were determined in plasma and isolated PMN cells in 46 patients in stable phase of COPD (past smokers, current smokers, and non-smokers), 24 healthy smokers and 37 healthy non-smokers. In the same samples concentrations of total (tCa) and ionised calcium (iCa) were determined, due to the antagonism of magnesium towards calcium. We found decreased biological active iMg in PMN compared to the group of healthy non-smokers (5.42, 1.98–17.31 μ mol/109 cells vs. 7.50, 3.27–15.15 μ mol/109 cells, p < 0.05). In the plasma and isolated PMN of the patients the ratio of total calcium/total magnesium (tCa/tMg) was significantly increased (2.89, 2.15–3.86 and 1.19, 0.07–9.87) compared to the group of healthy non-smokers (2.65, 2.19–3.44 and 0.67, 0.14–2.40, p < 0.05) and to the group of healthy smokers (2.58, 2.26–3.24 and 0.66, 0.14–2.85, p < 0.05). In the group of patients the concentration of tCa was significantly increased in all samples compared to the healthy group of non-smokers and healthy smokers. The results of univariant logistic regression analysis for smoking, concentration of tCa and ratio of tCa/tMg in PMN showed high odds ratio for COPD status. These results raise a possibility that intracellular polymorphonuclear value of magnesium could be a distinctive marker for COPD risk disclosure among smokers.
INTRODUCTION
In a large epidemiological investigation Britton et al. (Citation[1]) demonstrated correlation between impaired lung function, bronchial hyperreactivity and increased expectoration with low intake of magnesium in developed western countries. The negative effect of hypomagnesemia on the function of respiratory muscles, bronchial reactivity and pulmonary hypertension has been described (Citation[2]). Magnesium is important for the stabilisation of electrolyte homeostasis (Citation[3]) and participates in processes of the development of oxidative stress (Citation[4]). Oxidative stress is one of the important factors in chronic obstructive pulmonary disease (COPD). There is evidence for the influence of oxidants on inflammation (Citation[5]) as well as for the role of inflammation in the induction of oxidative stress. COPD is characterised by chronic local, and according to the latest data, systemic inflammation and oxidative stress (Citation[6]). Magnesium deficiency results in pro-inflammatory processes, increased phagocytosis, and increased formation of cytokines, reactive oxygen species and adhesive molecules (Citation[7], Citation[8], Citation[9], Citation[10], Citation[11], Citation[12]). The role of magnesium has also been described in the activation of endonucleases, i.e., apoptosis (Citation[13]). Furthermore, data exists on the possibly satisfactory effect of supplementing magnesium sulphate in standard therapy during the treatment of patients in exacerbation of COPD and asthma (Citation[14], Citation[15], Citation[16], Citation[17]).
The majority of investigations determined the concentration of magnesium in serum or plasma, which does not reflect the true state of the supply of the organism with magnesium, as only 1% of the magnesium is in blood plasma. With regard to cells it is considered that different types of cells have different kinetic interchange of magnesium and other electrolytes and it is still not completely clear which type of cell best reflects the status of magnesium in the organism (Citation[18], Citation[19], Citation[20]).
Because of the ever-increasing number of patients with COPD in the world and the aforementioned role of magnesium in oxidative processes and inflammations, in this study the concentrations of total and ionized magnesium were determined (tMg and iMg) in the plasma of patients with COPD. Namely, in the literature changes in the concentration of iMg have been described in some patients, which are responsible for regulation of various cellular processes, without changes in the concentration of tMg (Citation[21], Citation[22]). Because of the important role of polymorphonuclear (PMN) cells in the pathogenesis of COPD, the concentration of tMg and iMg were also determined in isolated cells.
As magnesium is a known antagonist toward calcium (Citation[23]) the concentration of total and ionised calcium (tCa and iCa) were determined, and the ratios of both forms of magnesium and calcium, in the same groups of patients and healthy subjects.
MATERIALS AND METHODS
Subjects
The examination was performed in 46 patients with stable COPD (13 women and 33 men, mean age 66 years, range 44–80 years), in 37 healthy subjects, non-smokers, (6 women and 31 men, mean age 53 years, range 40–72 years), and 24 healthy subjects smokers, (3 women and 21 man age 49 years, range 33–63 years) who served as the control group.
Approval for the study was given by the local Ethics Committee and all of the subjects gave their informed consent. The subjects completed a questionnaire which contained questions on smoking, blood pressure and possible other diseases.
For all subjects spirometry and standard laboratory tests in blood and urine were performed on the same day. Healthy subjects were included in the study on the basis of the values of standard blood and urine tests in a referent interval and normal finding of spirometry. Pulmonary function test of the group of healthy non-smokers and healthy smokers is shown in . Subjects with COPD, treated in the Clinical Hospital for Lung Diseases “Jordanovac,” Zagreb, had been in the stable phase of the disease for at least 3 months, without hospitalization and without change in therapy. The group of patients with COPD consisted of 11 non-smokers, 13 past-smokers and 22 current smokers, with forced expiratory volume (FEV1%) 30%–70% of the predicted value. The patients were considered past-smokers if they had not smoked a cigarette for 6 months. Current smokers were considered the patients who smoked at least two cigarettes a day, while non-smokers had never smoked on regular basis more than a cigarette a week.
Table 1 Pulmonary function test for patients with COPD, healthy control non-smokers and smokers
The diagnosis of COPD in non-smokers was established in patients who had reported obstructive ventilatory disorder with FEV1% below 70% of predicted value, for at least 6 months, and FEV1/FVC ratio less than 0.7. Bronchodilator response was negative which means that there was an increase in FEV1 less than 200 ml and/or 12% from baseline measured value. Pulmonary function test of the group of patients with COPD is shown in . According to GOLD classification patients were classified in grade 2 and 3 COPD. Therapy consisted of a combination of β2-agonist, anticholinergic and xanthic preparations, without corticosteroids. Subjects with diabetes, kidney disease, arterial hypertension, heart diseases, gastrointestinal diseases, endocrine diseases and asthma were excluded from the investigation.
Subjects take drugs such as calcium antagonists, diuretics, digoxin, laxatives, antibiotics, or who consumed more than 50 g alcohol daily, and those taking vitamin and mineral preparations, were also excluded from the investigation.
SAMPLES
The blood of patients with COPD and healthy volunteers was taken in the morning between 7–10 hours, and on the same day spirometry was performed. Plasma was obtained by taking 5 ml of blood in a plastic vacuum test-tube coated with anticoagulant Li-heparin 15 000 IU/L (Venosafe, Terumo) and centrifuged for 10 minutes at 3000 rpm. The plasma obtained was stored at −20°C until analysis.
The PMN obtained were modified by the method of isolation according to Bojum (Citation[24]): Blood is taken into a plastic test tube coated with anticoagulant Li-heparin 15000 IU/L (Venosafe, Terumo). Then 13 ml blood is mixed with 2.6 ml freshly prepared solution of dexstran (Dextran 500, 50 g/L) in a glass test-tube and left to stand for 60 min. at room temperature. Plasma rich in leukocytes is carefully layered on solution Ficoll-Paque-plus (< 0.12 EU/ml Amersham Pharmacia Biotech AB) ratio 2:1 and centrifuged at 400 g for 35 minutes. Because of the gradient of density during centrifugation PMN sediment on the bottom of the test tube and mononuclear cells is situated between the layer of Ficoll-Paque solution and the plasma. The PMN sediment is suspended in 1 ml of cold NaCl (9 g/L) and part of the erythrocytes removed. Finally, removal of erythrocytes is carried out successively by adding 3 ml cold distilled water, mixing for 45 seconds and then adding 3 ml of cold NaCl (18 g/L) and mixing for a further 45 seconds. The suspension is centrifuged for 10 min. at 500 g.
The procedure of removing the erythrocytes is performed at least three times. The PMN sediments are suspended in 1 ml of cold NaCl (9 g/L) and the cells counted on a haematological counter (Nihon Kohden Mek 5100). The remaining suspended cells are sonicated with an ultrasound homogenisator (Cole-Parmer 4710), holding the eppendorf test tube in ice, 3 × 30 seconds, in intervals of 45 seconds. Such a lysate of cells is divided into 0.5 ml in two eppendorf test tubes and stored at −20°C until analysis.
METHODS
The concentration of tMg and tCa was determined in plasma and PMN by the method of atomic absorption spectrophotometry (Atomic absorption spectrophotometer 2380, Perkin Elmer). The concentration of iMg and iCa in plasma and PMN was determined by the method of direct potentiometry on analyser Nova Ultra Stat Profile M (Nova Biomedical, USA Ltd.). Normalised values were taken at a temperature of 37°C and pH 7.4.
Procedure for spirometry
Before starting the measurements the patient's height and weight was measured without shoes. The test was performed in a seated or standing position. After applying noseclips, the subject was instructed to take a full inspiration, hold it briefly then exhale through a mouthpiece into the spirometer as forcefully and completely as possible, for at least 6 seconds. The test was repeated a minimum of three times, maximum eight times, until two reproducible efforts were obtained. The two largest FVCs and FEV1s had to show < 5% variability. After the spirometry measurement, according to standard procedure, the patient inhaled 400 μ g of salbutamol from the MDI.
After 30 minutes, spirometry was repeated three times and the highest FEV1 value recorded. Before testing, regularly prescribed bronchodilators were withheld for 6 hours for inhaled short acting β2-agonist, 12 hours for long acting β2-agonist, 6 hours for inhaled anticholinergics, and 12 hours for short acting theophylline preparations and 24 hours for long acting theophylline preparations. Bronchodilatatory test was considered negative if the increase of FEV1 after salbutamol was less than 200 ml and/or 12% before testing. Reference values were used according to ECCS, 1983 (Citation[25]).
STATISTICAL METHODS
Mean values of parameters for each group of subjects are presented in medians and ranges. Differences between the examined and control groups were tested by non-parametric Mann-Whitney U test. Univariant logistic regression analysis was made, and when normality tests failed, a logarithmic transformation was applied to the data. A value of p < 0.05 was taken significant.
RESULTS
Median, mean and analogous ranges in the concentration of magnesium and calcium, and their ratio in the plasma and PMN of the subjects are presented in . The concentration of iMg and tMg in the plasma of patients did not differ significantly in relation to the control group of healthy non-smokers and healthy smokers. However, the concentration of iMg was significantly lower in PMN patients with COPD in relation to the control group of healthy non-smokers (median 5.42 μ mol/109 cells, range 1.98–17.31 vs. median 7.50 μ mol/109 cells, range 3.27–15.15, p < 0.05).
Table 2 Median, mean, and ranges for concentration of total and ionized magnesium (tMg and iMg) and total and ionized calcium (tCa and iCa) and their ratios in COPD patients and control subjects of healthy non-smokers and healthy smokers
A statistically significant increase in the concentration of tCa was found in plasma and PMN of patients with COPD (median 2.42, range 2.20–2.64 mmol/L, and median 5.26, range 0.24–50.94 μ mol/109 cells, p < 0.05) compared to the control group of healthy non-smokers (median 2.31, range 2.15–3.32 mmol/L, and median 3.03, range 0.47–16.67 μmol/109 cells), and healthy smokers (median 2.28, range 2.10–2.50 mmol/L and median 2.72 μ mol/109 cells, range 0.62–16.39). The ratios of tCa/tMg in plasma and PMN were significantly greater (p < 0.05) in patients with COPD (median 2.89, range 2.15–3.86 and median 1.19, range 0.07–9.87) compared to the control group of healthy non-smokers (median 2.65, range 2.19–3.44 and median 0.67, range 0.14–2.40) and healthy smokers (median 2.58, range 2.26–3.24 and median 0.66, range 0.14–2.85).
It can be concluded that in PMN of the patients with COPD a change occurred in the concentration of iMg compared to the control group of non-smokers, while there was no change compared to the control group of smokers. In addition, the concentrations of tMg and iMg in the plasma of patients with COPD were not changed compared to both healthy group non-smokers and smokers. There was a marked increase in the concentration of tCa in the plasma and PMN in patients with COPD in relation to the control group of non-smokers and smokers, with no change in its ionising form.
The ratios of tCa/tMg in plasma and PMN of patients with COPD were greater in relation to the control group of non-smokers and smokers, which indicates relative tMg deficiency with regard to the concentration of tCa. We used multiple stepwise regression analysis, in which FEV1 % was included as a dependent variable and ratios tCa/tMg as independent variables in plasma and PMN of the patients with COPD. We found statistical significance of p = 0.041 and negative coefficient regression R = −0.483 for ratio tCa/tMg in the patients plasma.
There is also no statistically significant difference in any parameters between smokers (past smokers and current smokers) and non-smokers in the group of patients with COPD. Because of that but also because of the fact that the number of non-smokers was low, a group of patients with COPD, which included past smokers, current smokers and non-smokers, was used in order to determine a statistically significant difference and for univariant logistic regression analysis. Further on, there is not statistically significant difference between the groups of healthy smokers and healthy non-smokers in any parameter and both groups were used for univariant logistic regression analysis. The results of the univariant logistic regression analysis for smoking, concentration of tCa and the ratio of tCa/tMg in PMN as independent variable showed high odds ratio for COPD status (). Odds ratio for smoking (OR = 4.765, 95%CI 2.012–11.285, p < 0.05) confirms the influence of smoking on the development of COPD, as proved earlier.
Table 3 The results of univariant logistic regression analysis for smoking, concentration of tCa and ratio of tCa/tMg in PMN as independent variable and for COPD status as dependent variable
DISCUSSION
In this study the concentration of tMg and iMg in PMN was determined for the first time in patients with COPD in a stabile phase. Because of the aforementioned antagonistic effect of Mg toward Ca in many cellular processes, the concentration of tCa and iCa was determined and the ratios of tCa/tMg and iCa/iMg in the same samples.
For diagnosis and determination of the degree and prognosis of COPD, according to GOLD standards (Citation[26]) parameters such as spirometric parameter FEV1 and blood gas analysis parameter pO2 are generally used. Many inflammatory cells, mediators and enzymes are involved in the disease process.
COPD is characterized by local chronic inflammation involving the airways, parenchyma and blood vessels of the lungs, in which macrophages, T-lymphocytes (mainly CD8+) and neutrophils occur in an increasing number in different parts of the lungs. More recently there has been an increasing amount of data that indicates that COPD is not only a disease of the lungs but also a systemic inflammatory disease that includes persistent systemic oxidative stress, activation of inflammatory cells and increased concentration of primary proinflammatory and also antiinflammatory cytokines. The main cause of the systemic inflammation has yet to be explained, although one of the candidates—causal agents—is probably systemic hypoxy (Citation[27]).
The activated inflammatory cells in patients with COPD release different inflammatory mediators, leukotrien (LTB4), interleukin 8, TNF-alfa and others, which can damage the lungs and/or maintain the inflammatory reaction, characteristic with regard to the large number of neutrophils (Citation[28]). Oxidative stress causes oxidative inactivation of antiproteinases, damage to airways, and increased sequestration of neutrophils in pulmonary circulation and expression of the gene for proinflammatory mediators (Citation[29]).
Burnet et al. (Citation[30]) demonstrated that the neutrophils in patients with COPD show increased chemotaxis and extracellular proteolysis. Furthermore, Noguera et al. (Citation[31]), showed that the neutrophils in patients with COPD create more reactive oxygenic compounds than the neutrophils of healthy smokers and non-smokers, and show increased expression of MAC-1 (CD 11b) of adhesive molecules. Other disorders in neutrophils include impaired regulation of G-protein type Gas, which regulates expression of adhesive molecules and commencement of cascade reaction for activation of NADPH-oxidase (Citation[32], Citation[33]).
Mg has an important role as cofactor in numerous biochemical processes in the cell, including processes in the occurrence of oxidative stress and regulation of inflammatory response (Citation[4], Citation[11]). It has been demonstrated on different animal models that the lack of magnesium results in an inadequate inflammation response (Citation[12]). High concentrations of Mg in vitro reduce proinflammatory processes by influencing activation of leukocytes in the process of phagocytosis, thereby reducing the formation of free radicals (Citation[35]). The increase of calcium in the cells is the result of the activation of leukocytes and activity of calcium-dependent protein kinase.
Ca is furthermore an important “second messenger” in the signal processes of oxidative stress in leukocytes and is involved in the formation of certain eikosanoids. The formation, for example, of leukotriens through activation of phospholypase A2 and 5-lipoxygenase depends on calcium, and magnesium is an inhibitor of phospholypase A2 and a natural blocker of calcium-canals. Prostaglandin D2 and leukotriens are furthermore bronchoconstrictors with an influence on the secretion of mucous (Citation[9]). Mg is an inhibitor of enzyme NADPH-oxidase, present on membranes of neutrophils, enzymes that catalyze the first reaction in the occurrence of oxidative stress (Citation[33]).
As Mg acts as an antagonist toward calcium ions in many physiological and pathophysiological processes it can be assumed that extracellular Mg can decrease activation of leukocytes by its antagonistic effect on Ca (Citation[35]). The majority of studies published in the literature have determined the concentration of tMg in the serum and erythrocytes of patients with asthma. Good correlation has been determined between FEV1 and the concentration of tMg in erythrocytes (Citation[36]). A decreased content of tMg was demonstrated in PMN patients between asthmatic attacks (Citation[37]).
Furthermore, numerous studies have shown better response to therapy with beta2-agonist with simultaneous application of intravenous magnesium-sulphate in the treatment of asthma (Citation[38]) and few studies on patients with COPD (Citation[17]). Skorodin et al. (Citation[39]) for example examined the effect of magnesium sulphate as a supplement in therapy with beta2-agonist and established better therapeutic effect in patients with exacerbation of COPD. In one published study decreased Mg in the serum of a patient with COPD was connected with the application of a diuretic (Citation[40]) and in another study with decreased activity of antioxidative enzymes in erythrocytes (Citation[41]).
There were no changes in the concentration of tMg and iMg in the plasma of patients with COPD, while a statistically significantly increased ratio tCa/tMg was determined compared to the control group of healthy non-smokers and healthy smokers. In PMN patients with COPD the concentration of physiologically active iMg was decreased compared to control group of healthy non-smokers, the concentration of tCa and the ratio of tCa/tMg increased, which together indicates relative tMg deficiency and suggest the conclusion of intensified activity of Ca. When comparing the data for iMg and tMg, the data for iMg are higher than those for tMg in PMN, and they are the same for iCa and tCa.
Concentration of tCa or tMg must be higher than the concentration of the subfraction. It is possible that the discrepancies in the PMN levels of Mg and Ca arose from calibration artefacts (PMN compartment is different from plasma compartment). The values measured for tCa and tMg in the plasma are higher than those for iCa and iMg indicating the absence of analytical art-efacts. This conclusion is also backed by the results of univariant logistic regression analysis where high odds ratio for concentration of tCa and ratio tCa/tMg in PMN was obtained.
It also confirmed the influence of smoking on the development of COPD, which had been proved earlier. Smoking is considered the major risk factor for development of COPD and accounts for 90% of the cumulative risk. In this work current smokers and past smokers represent 76% of the group of the patients with COPD. As not all smokers develop COPD, genetic factors are likely to modify the risk of developing COPD.
On the basis of these results and the role of PMN in the pathogenesis of COPD it can be concluded that decreased concentration of iMg and increased ratio of tCa/tMg (i.e., relative deficit of tMg in relation to tCa) results in intensified formation of free radicals and the occurrence of proinflammatory processes, due to the inadequate activation of leukocytes, which is characteristic of COPD. These results raise a possibility that intracellular polymorphonuclear value of magnesium could be a distinctive marker for COPD risk disclosure among smokers, what should be investigated on bigger number of COPD patients.
The drugs used today in the treatment of stable COPD do not slow down the significant progression of the disease (Citation[42]). Based on the results of this study it can be hypothesized that the supplementation of magnesium salts to standard therapy, in patients with stable COPD, can improve the symptoms and possibly reduce the number of exacerbations of the disease. The hypothesis is justified by the participation of magnesium in the defense reactions of oxidative stress and regulation of the activation of leukocytes, and the relaxing effect on the smooth bronchial muscles.
Asthma and COPD per se may induce Mg depletion related to a dysregulation of the control mechanism of Mg status. When chronic primary Mg deficiency coexists with COPD, non-toxic nutritional Mg therapy may palliate this coexistent Mg deficiency. It is difficult to obtain at the same time high efficacy and high safety level of pharmacological Mg therapy for COPD. Combination of palliating nutritional Mg therapy and of beta2-mimetics may be beneficial and remains non-toxic (Citation[43]). In this respect there is a need for a further longitudinal study and continued monitoring in order to determine how such therapy with magnesium salts correlate with FEV1.
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