INTRODUCTION

Heart failure (HF) is a serious problem in contemporary cardiology, that is why a lot of research has been carried out to discover the factors having prognostic value, possibly opening new therapeutic possibilities. A substantial part of those studies concerns the disturbances of the oxidative-antioxidative balance, as mechanism of that disease’s progression. Still, however, there is no agreement as to the role of oxidative stress in the development of HF [1, 2]. Hypoxia accompanying the diseases with tissue perfusion disturbances in their course is, as is known, a factor causing the generation of reactive oxygen species (ROS), which encourages for examining the oxidation condition of patients with HF [3, 4].

Aim of the study: determination of oxidation status of patients with idiopathic cardiac insufficiency (heart failure), on the basis of selected biochemical parameters.

MATERIAL AND METHODS

The study group consisted of 37 patients of the average age of 42.3±12.3 years, suffering from idiopathic or inflammatory heart failure. The patients have been qualified as degree II/III in accordance with NYHA scale. In the course of 6-month observation they were clinically stable, which got expressed by the fact that the NYHA scale degree was maintained. The patients were treated in accordance with the classical HF treatment scheme, comprising β-blocker inhibitor of angiotensin convertase, aldosterone antagonist, and palliative treatment with a diuretic. Intensity of lipid peroxidation was measured fluorometrically as thiobarbituric acid-reactive substances (TBARS) in blood plasma according to Ohkawa [5] after reaction with sodium dodecyl sulfate, acetic acid, 2-thiobarbituric acid and butanol-pyridine mixture. The butanol-pyridine layer was measured fluorometrically at 552 nm (515 nm Excitation) by means of spectrofluorimeter Shimadzu (Rydalmere, Australia). Tetraethoxypropane was used as the standard. TBARS values were expressed as malondialdehyde (MDA) equivalents in µmol/l of plasma or nmol/gHb in erythrocytes.

The activity of SOD was determined by the Oyanagui [6] method. Enzymatic activity was expressed in nitric unit (NU) per mg of haemoglobin (Hb) or ml of blood plasma. One nitric unit (1 NU) corresponds 50% of inhibition by SOD of nitric ion production in this method. In the blood plasma SOD isoenzymes (SOD-Mn and ZnCu-SOD) were also determined using KCN as the inhibitor of the ZnCu-SOD isoenzyme. The level of anti-oxLDL antibodies (oxLDL-Ab) was measured in serum by ELISA method (BIOMEDICA).

Statistical analyses

All the measurements have been performed twice over the 6-month period. Statistical analysis has been performed using the Wilkoxon test.

RESULTS

Clinical characteristic of studied patients are shown in table I. There were no statistically significant difference between studied groups.

A statistically significant increase of anty-oxLDL level and MDA (µmol/l) level in plasma has been demonstrated over the course of the 6-month observation. The results are presented in table II.

No statistically significant differences have been found for the activity of total SOD, as well as SOD-CuZn and SOD-Mn in plasma of studied patients. The results are presented in table III.

DISCUSSION

We are incessantly subject to reactive oxygen species generation (superoxide anion, its protonated form – hydroxyl radical, hydroperoxide radical, as well as the hydrogen peroxide H2O2 and singlet oxygen 1O2, not being free radicals), yet we have at our disposal a variety of enzymatic antioxidants (superoxide dismutase SOD, glutathione peroxidase GPX, catalase CAT), as well as non-enzymatic ones (hydrophilic ones: albumin, glutathione, ascorbate, mannitol, uric acid, metallothionins, cysteine, neopterine, and melatonin. Hydrophobic ones: tocopherols, coenzyme Q, bilirubin, xanthophils, derivatives of oestrone and oestradiol) [7]. If, however, for any reason the antioxidative system is inefficient, or the production of ROS is increased, the oxidative-antioxidative balance is disturbed, thus oxidative stress occurs, which may lead to dysfunction of numerous organs [8]. What is postulated is the influence of ROS upon contractility of myocardium. ROS reduce the contractile activity of cardio-myocytes due to reactions with lipids or membrane proteins, the diminish contractility of cardiac muscle reduces tissue perfusion, which results in the occurrence of hypoxia, which increases the ROS generation. Thus, a classical mechanism of positive feedback is manifested, characteristic for many pathological processes [9].

Several mechanisms of ROS generation are possible, in condition of decrease oxygen supply. One of them is the inhibition of ATP re-synthesis, resulting in increased production of uric acid, which is accompanied by the generation of superoxide anion by xanthine oxidase [10]. Another possible mechanism is connected with the respiratory chain and the intensification of single-electron oxygen reduction in inhibition of oxidative phosphorylation. A third mechanism is connected with increased concentration of catecholamines, which cause in the heart the so-called oxygen wasting effect, consisting of intensification of oxygen reduction in the respiratory chain, without the generation of ATP [7].

We have observed no changes in SOD activity that may indicate the inefficiency of the antioxidant system. Many research reports quote the decreased activity of SOD in the condition of hypoxia or ischaemia [11]. Our results differ from the studies by Yucel et al. [12] who report reduction of SOD activity in patients with HF. This difference may be due to different NYHA functional class , which would indicate the possibility of disease progression with the SOD inefficiency. As it is known, SOD is rendered inactive by its product H2O2. In order to maintain the activity, it requires the enzymes decomposing hydrogen peroxide [13].

Those enzymes are catalase and glutathione peroxidase. Catalase is present in the form of 2 isoenzymes: the typical one (T) in cytosol, and atypical one (A) in peroxisomes. It is absent, however, in mitochondria. There, the protective role for SOD is played by glutathione peroxidase, the activity of which is lesser however, and it depends upon glutathione [14]. That is why, taking into consideration also the rate of ROS generation in cytosol and mitochondria, Mn-SOD seems to be more prone to inactivation by H2O2. However, on the basis of the data we collected (intitially and finally lower activity of CuZn-SOD than of Mn-SOD) and data from the research conducted by Jacheć et al. [15], who state that the activity of CuZn-SOD dropped to zero, while the activity of Mn-SOD was maintained in patients with NYHA functional class IV, thus it may by supposed that dominating is the generation of ROS connected with xanthine oxidase, and the oxidative-antioxidative balance in cytosol is had been disrupted earlier.

The role of manganese-dependent isoenzyme may be testified by genetic studies, which had proven that in the promotor sequence of the gene for Mn-SOD, located on chromosome 6, the polymorphism of that sequence exists, and it turned out that certain alleles bind are connected with increased risk of heart failure with the background of dilated cardiomyopathy [16]. An indirect proof of the inefficiency of antioxidative system is the increase of concentration of lipid peroxidation markers, among them the concentrations of MDA and anti-oxLDL antibodies we determined. As a result of oxidative stress, from the native form of LDL (low density lipoproteins), oxLDL (oxidised LDL) particles are generated, the pathogenic role of which has been demonstrated in many diseases of the circulatory system, the most important of those diseases being atheromatosis, in many case being the underlying cause of further diseases affecting human organism. In contrast to oxLDL, native LDL does not seem to be a harmful factor [17]. nLDL i
s accumulated in the walls of blood vessels, under the influence of oxidative stress the polyunsaturated fatty acids get transformed into lipid peroxides, with the formation of oxLDL. In serum, we determined the level of anti-oxLDL antibodies, which is directly proportional to the concentration of oxLD. Besides the undisputable role in the process of atherosclerosis generation, the harmful activity of oxLDL is also based upon its ability to stimulate free radicals, particularly O2- [3], speeding-up the atherosclerosis process, de-stabilizing of atheromatous plaque, pro-coagulation and cytotoxic activity [18, 19]. In cardiac insufficiency, so far there has been no clear proof for the pathogenetic role of oxLDL, we demonstrated, however, a significant increase of anti-oxLDL antibody level in the studied patients with heart failure. The level of oxLDL in blood serum is a marker of oxidative stress, it has also been recognised as a prognostic indicator of congestive heart failure – CHF. A high level of oxLDL correlates positively with incidence and mortality in patients with CHF (congestive heart failure) and is an independent mortality risk factor. In patients with severe form of CHF the increase of oxLDL level is substantial [3, 19].

The results of the study indicate that no changes in SOD activity and increase in lipid peroxidation products level may lead to generation of oxidative stress may play an important role in the pathogenesis of heart failure.

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LITERATURE

1.     Ferrari R, Guardigli G, Mele D, Percoco GF, Ceconi C, Curello S: Oxidative stress during myocardial ischaemia and heart failure. Curr Pharm Des 2004; 10 (14): 1699-1711.

2.     Trachtenberg BH, Hare JM: Biomarkers of oxidative stress in heart failure. Heart Fail Clin 2009; 5 (4): 561-577.

3.     Radovanovic S, Krotin M, Simic DV et al.: Markers of oxidative damage in chronic heart failure: role in disease progression. Redox Rep 2008; 13 (3): 109-116.

4.     Charniot JC, Vignat N, Albertini JP et al.: Oxidative stress in patients with acute heart failure. Rejuvenation Res 2008; 11 (2): 393-398.

5.     Ohkawa H, Ohisi N, Yagi K: Assay for peroxides in animal tissue by thiobarbituric acid reaction. Annal Biochem 1979; 95: 351-358.

6.     Oyanagui Y: Reevaluation of assay methods and establishment of kit for superoxide dismutase activity, Anal Biochem 1984; 142: 290-296.

7.     Giordano FJ: Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest 2005; 115 (3): 500-508.

8.     Seddon M, Looi YH, Shah AM: Oxidative stress and redox signaling in cardiac hypertrophy and heart failure. Heart 2007; 93 (8): 903-907.

9.     Zhang M, Shah AM: Role of reactive oxygen species in myocardial remodeling. Curr Heart Fail Rep 2007; 4 (1): 26-30.

10.     Bergamini C, Cicoira M, Rossi A, Vassanelli C: Oxidative stress and hyperuricaemia: pathophysiology, clinical relevance and therapeutic implications in chronic heart failure. Eur J Heart Fail 2009; 11 (5): 444-452.

11.     Alcaino H, Greig D, Chiong M et al.: Serum uric acid correlates with extracellular superoxide dismutase activity in patients with chronic heart failure. Eur J Heart Fail 2008; 10 (7): 646-651.

12.     Yücel D, Aydoğdu S, Cehreli S et al.: Increased oxidative stress in dilated cardiomyophatic heart failure. Clin Chem 1998; 44 (1): 148-154.

13.     Amir O, Paz H, Rogowski O et al.: Serum oxidative stress level correlates with clinical parameters in chronic systolic heart failure patients. Clin Cardiol 2009; 32 (4): 199-203.

14.     Indik JH, Goldman S, Gaballa MA: Oxidative stress contributes to vascular endothelial dysfunction in heart failure. Am J Physiol Heart Circ Physiol 2001; 281 (4): 1767-1770.

15.     Jacheć W, Poloński L, Kuśnierz B: Superoxide dismutase isoenzymes activity changes in the plasma of heart failure patients. Med Sci Monit 1998; 4 (4): 646-650.

16.     Koba S, Gao Z, Sinoway LI: Oxidative stress and the muscle reflex in heart failure. J Physiol 2009; 587 (21): 5227-5237.

17.     Assadpoor-Piranfar M, Pordal AH, Beyranvand MR: Measurement of oxidized low-density lipoprotein and superoxide dismutase activity in patients with hypertension. Arch Iran Med 2009; 12 (2): 116-120.

18.     Banfi C, Brioschi M, Barcella S et al.: Oxidized proteins in plasma of patients with heart failure: role in endothelial damage. Eur J Heart Fail 2008; 10 (3): 244-251.

19.     Tsutsui T, Tsutamoto T, Wada A et al.: Plasma oxidized low-density lipoprotein as prognostic predictor in patients with chronic congestive heart failure. J Am Coll Cardiol 2002; 39 (6): 957-962.

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*ADRES DO KORESPONDENCJI:

Ewa Romuk
Zakład Biochemii Ogólnej, Katedra Biochemii
Śląski Uniwersytet Medyczny
41-808 Zabrze, ul. Jordana 19
tel./fax: 32 272 23 18
e-mail: eromuk@gmail.com

Pracę nadesłano: 13.11.2010 r.
Przyjęto do druku: 04.04.2011 r.

Praca powstała na podstawie referatu wygłoszonego podczas XVII Zjazdu Polskiego Towarzystwa Diagnostyki Laboratoryjnej w Wiśle, 14-17.09.2010 r.