FREE RADICAL OXIDATION IN THE MODELS OF ISCHEMIA
Kemerovo State Medical Academy,
Kemerovo, Russia,
Moscow State Medicine and Dentistry University named after A.I. Evdokimov,
Moscow, Russia
Currently, many authors consider the uncontrolled activation of free radical processes as one of the main pathogenetic factors of structural and functional injuries to organs and tissues in primary chronical pancreatitis (PCP) and myocardial infraction (MI) accompanied by ischemic and inflammatory processes [1, 3]. The limited level of lipid peroxidation is necessary for supporting the vital activities of the cells. It is one of the normal ways of catabolism, whereas increasing level of lipid peroxidation makes destructive influence on the cells [4].
Ischemia creates the paradoxial situation, when decreasing oxygen level leads to increase in cytotoxic active forms of the oxygen. Activation of the processes of lipid peroxidation in ischemia is combined with development of insufficiency of antioxidant system (AOS), and decrease in level of oxidative stress is accompanied by simultaneous increase in antioxidant activity of the blood [5].
Despite of all multiple causes of PCP, pathogenesis is characterized by increasing pressure in the ductal system of the pancreas, reflux in Wirsung canal and (or) duodenal contents. The role of ischemia in pathogenesis of pancreatic diseases is insufficiently described. For example, the pancreas creates ischemia with worsening course of pancreatitis [6]. The range of the studies showed that ischemia causes the insignificant influence on the pancreas, although other authors see the inverse relationship [7, 8]. It is necessary that all types of PCP are associated with the noticeable role of the changes in the microcirculation system leading to hypoxia of the pancreatic cells [9].
As for pathogenesis of coronary heart disease, free radical oxidation is considered as a cause and a consequence of this disease, because the processes of lipid peroxidation play the leading role in stress myocardial injury and development of atherosclerosis [10].
It is supposed that activation of lipid peroxidation in the region of ischemia is associated with peripheral development of boundary prooxidant region, which is different from other regions of MI in view of partial restoration or blood flow and oxygen delivery from one side, and lower oxygen utilization by cardiomyocytes from other side [10].
Some authors suppose that free radical oxidation has its own role in development of postischemic dysfunction [11]. Moreover, oxidative stress is considered as one of the mechanisms of development of apoptosis, which is often observed in hibernation. Postinfarction sclerosis significantly decreases energetic resources even in uninjured regions of the heart. It increases the cellular sensitivity to acute metabolic stress [12].
The general pathogenetic links of development and progression of PCP, coronary heart disease and chronical cardiac insufficiency include oxidative stress [13-15].
MATERIALS AND METHODS
The experimental model of chronical pancreatitis was developed at the first stage of the examination. Experimental chronical pancreatitis was performed for 175 white male rats (Wistar line), the weight of 200-220 grams in concordance with the recommendations of the International Committee of Science of Laboratory Animals approved by WHO. The experimental model and the course of pancreatitis were studied according to G. Sparmann et al. (1997) [16] and J. Merkord et al. (1997, 1998, 1999) [17]. 0.6 % of DBTC solution inducing PCP was introduced into the caudal vein (6 mg/kg). DBTC stimulated toxic necrosis in biliopancreatic epithelium of the ducts. Intense periductal interstitial fibrosis and active inflammatory process in the pancreas developed after 28 days. 102 (58 %) animals were lost during the process of modelling of chronical pancreatitis. Creation of modelled chronical pancreatitis was completed in 28-30 days after beginning of the experiment.
The analysis included the static (contents of the products of peroxide oxidation and activity of superoxide dismutase (SOD) in tissues in vivo) and dynamic changes (varying levels of the products of peroxide oxidation and SOD activity in vitro in incubation of liver homogenates and the pancreas), clinical and biochemical examinations: malondialdehyde (MDA), diene conjugates (DC), antioxidant activity (AOA), ceruleoplasmin (CP) in the serum, SOD and catalase (CAT) in red blood cells.
The second stage of the study was oriented to investigation of the clinical factors and laboratory values in the patents with MI and PCP.
The first group included 135 patients with PCP of alcohol origin of various severity (the age of 21-70) with need for surgical intervention with resection of the pancreas and its ducts. The exclusion criteria were the age of 20 and older than 70, secondary (biliary) chronical pancreatitis, iatrogenic lesions of extrahepatic bile ducts, pancreatic injuries.
All examinations were conducted after acquisition of written consent from the patients and corresponded to the ethical standards of Helsinki declare (2000).
It was found that PCP more often developed in men of young and middle working age (31-50 years). It gives the important social significance to the problem. 135 cases (100 %) were associated with alcohol abuse (more than 250-300 ml of vodka daily within 7-15 years and more). Smoking as a burdening factor of PCP origin was identified in 119 (88.1 %) cases.
The second study group included 594 patients (37 women, 557 men) admitted to the department of acute coronary pathology within 24 hours after development of primary Q-wave MI. The mean age of the patients was 51.8 ± 1.1 (35-68). After development of MI the examination was conducted within the first 24 hours, 3rd day and 15th day after appearance of MI.
The diagnosis of MI was based on the criteria recommended by the WHO expert group. The criteria include the typical picture of the disease (pain syndrome in the chest lasting for not less than 30 minutes), the specific changes in ECG (ST segment elevation > 0.1 mV at least in one or two standard precordial abductions) and time course of activity MV fraction of creatine phosphokinase.
The clinical features of the patients with MI are presented in the table 1.
Table 1
| The characteristics of the patients with myocardial infarction at various stages of the examination |
|
Signs |
Terms | ||
| 1st day, n = 343 | 3rd day, n = 142 | 15th day, n = 454 | |
|
- Anterior myocardial infarction, n (%) |
177 (51.6) | 75 (52.8) | 254 (55.9) |
|
- Inferior myocardial infarction, n (%) |
166 (48.4) | 67 (47.2) | 200 (44.1) |
| Ventricular dysrhythmia, n (%) | 139 (40.5) | 35 (24.6) | 133 (29.3) |
| Cardiac insufficiency, n (%) | 126 (36.7) | 48 (33.8) | 137 (30.2) |
| Stenocardia (in anamnesis), n (%) | 67 (19.5) | 15 (10.6) | 80 (17.6) |
| AH, n (%) | 135 (39.3) | 44 (31) | 183 (40.3) |
| Diabetes mellitus, n (%) | 35 (10.2) | 4 (2.8) | 50 (11.0) |
| Repeated MI, n (%) | - | - | - |
| Relapse IM, n (%) | - | 2 (1.4) | 7 (1.5) |
| Fatal outcome, n (%) | 5 (1.5) | - | 5 (1.1) |
|
Note: the brackets show the percent number of the patients in relation to a number of the patients at each stage of the examination. |
|||
The methods of estimation of lipid peroxidation status
The clinical estimation of prooxidant and antioxidant status was conducted with the following values: DC – intermediate products of lipid peroxidation; MDA; total antioxidant activity of blood plasma, which was estimated with accumulation of the end products of lipid peroxidation in the modelled system – MDA; total antioxidant activity, which was estimated with degrees of inhibition of ascorbate- and ferro-induced oxidation of Tween-80 to MDA; the integral value – the summed AOA in blood plasma; ceruloplasmin (CP); CAT activity in red blood cells; SOD activity. The factor of antioxidative state was used for estimation of the balance between the processes of lipid peroxidation and activity of antioxidant system. The calculations were done with the formula: FAS = [MDA] ´ [DC] / [SOD] ´ [CAT]. The results were expressed in conventional units. The photometric measurements were conducted with the kinetic spectrophotometer Ultraspec Plus UV/Vis (Pharmacia LKB). The statistical analysis was conducted with Statistica 6.0 and Student and Mann-Whitney tests. For description of the signs with normal distribution we used the mean (M) with indication of the error in the mean (m). Differences were considered as statistically significant, if p value was less than 0.05.
RESULTS AND DISCUSSION
The tables 2 and 3 demonstrate the results of the examinations of the blood and homogenates in the pancreas and the liver of the experimental rats with modelled chronic pancreatitis.
The data (the table 2) shows that development of PCP is accompanied by development of oxidative stress.
Table 2
|
The values of oxidative disorders determined by development of experimental chronic pancreatitis in the blood of the animals |
|
Groups of animals |
Serum |
Red blood cells |
||||
|
MDA, µm/l |
DC, E232/ml |
CP, mg/100 ml |
AOA, % |
CAT, μm/min/ml |
SOD, U/ml |
|
|
Control (n = 8) |
6.1 ± 0.3 | 3.7 ± 0.1 | 28.7 ± 1.9 | 41.9 ± 2.1 | 13.5 ± 0.4 | 1533 ± 60.3 |
|
Chronic pancreatitis (n = 8) |
33.5 ± 1.2** |
4.8 ± 0.2 | 32.9 ± 1.4 |
16.8 ± 0.8** |
18.5 ± 1.5 | 856 ± 32.6* |
|
Note: authenticity of distinctions of values between healthy animals and experimental pancreatitis was found at *p < 0.05; ** p < 0.01. |
||||||
The data of the correlation analysis confirmed the identified orientation of the oxidative processes. So, the high degree of direct correlation was found between activity of catalase and the level of MDA in the blood of the animals with PCP: r = 0.857 (p < 0.01). The inverse statistically significant relationship was identified between activity of SOD and the level of the primary products of lipid peroxidation and DC: r = -0.929 with p < 0.002. Such relationship was not found in the intact animals (the control group). The healthy animals demonstrated other relationship between the examined vales: the direct relationship between two key enzymes of antioxidant protection – SOD and CAT (r = 0.812, p < 0.05), the inverse relationship – between the level of the primary products of lipid peroxidation – DC and the level of plasma antioxidant – ceruloplasmin (r = -0/986, p < 0.005). It is evident that development of pathology in the blood of the animals is accompanied by the inverse correlation between the links of oxidative and antioxidative processes. Increasing pool of the peroxile products of the blood leads to development of irreversible changes presenting the basis of fragmentation and destruction of membranes and cell death. The necessity of estimation of the rate of changes in levels of lipid peroxidation products in the examined homogenates is determined by the fact that the level of the products of lipid peroxidation in tissues does not always give adequate picture of changes in the correlation between pro- and antioxidant activity. Moreover, there is an opinion that estimation of patterns of accumulation of the lipid peroxidation products in tissue homogenates during incubation allows finding the changes in intensity of lipid peroxidation without appearance of changes in levels of lipid peroxidation products in tissues.
The examination of the hepatic and pancreatic tissues (the table 3) in the animals with modelled chronic pancreatitis showed the high level of the secondary products of lipid peroxidation: 27 % increase in the liver and 23 % increase in the pancreas in the control group. The activity of the main enzyme of antioxidant protection (SOD) increased in the pancreas (by 10 %) and in the liver (by 30 %) in the control group.
Table 3
| The changes in the markers of oxidative stress in experimental chronic pancreatitis in pancreas and liver of the animals |
|
Groups of animals |
Pancreas | Liver | ||
| MDA, nmol/ml tissue | SOD, rel. units/g tissue | MDA, nmol/ml tissue |
SOD, rel. units/g tissue |
|
|
Control (n = 10) |
38.9 ± 2.5 | 0.303 ± 0.041 | 45.8 ± 4.5 | 0.236 ± 0.039 |
|
Ñhronic pancreatitis (n = 10) |
47.7 ± 3.2* | 0.335 ± 0.045 | 71.6 ± 5.8* | 0.292 ± 0.032* |
|
Note: authenticity of distinctions of values between healthy animals and experimental pancreatitis was found at *p < 0.05. |
||||
Therefore, disorders in oxidant-antioxidant system present the important pathogenetic link in development and progression of PCP and its complications.
Lipid peroxidation status in patients with PCP
The patients with chronic pancreatitis were examined for the main markers of oxidative stress (the table 4). It was found that the amount of MDA was higher than the control values in the blood of the patients with PCP (by 66.1 % on average, p < 0.01). The activity of antioxidant protection enzymes is significantly reduced: erythrocytic CAT was 28 % lower than the control level (p < 0.01), SOD activity – 24 % lower than the control indices in the healthy individuals. Along with accumulation of the oxidation products, the predominance of the oxidative processes in the patients with PCP confirms the increase in total oxidative activity before surgery. The decrease in activity of the main enzymes of AOS with simultaneous decrease in total antioxidant activity determines the decrease in AOS reserves and incapacity of adequate response of such system. Moreover, the level of ceruloplasmin was 37.6 % higher than the control level. The identified increase in the level of ceruloplasmin is determined by the fact that, besides antioxidant function in blood serum, ceruloplasmin is a protein of acute phase with increasing synthesis in inflammatory processes. Its increase in blood serum primarily testifies the activity of a pathologic process and is considered as a compensatory response of the body.
Table 4
| The values of lipid peroxidation in patients with primary chronic pancreatitis |
|
Index |
Healthy (n = 30) |
Primary chronic pancreatitis (n = 135) |
P |
|
DOA, % |
4.6 ± 0.8 | 6.3 ± 1.0 | > 0.05 |
|
MDA, nmol/ml |
5.6 ± 0.9 | 9.3 ± 1.5 | < 0.01 |
|
LHP, c.u. |
3.5 ± 0.75 | 3.3 ± 0.54 | > 0.05 |
|
Erythrocytic SOD, % |
45.0 ± 3.0 | 34.2 ± 2.7 | < 0.05 |
|
Erythrocytic CAT, % |
71.5 ± 6.5 | 51.5 ± 5.9 | < 0.01 |
| AOA, % | 8.5 ± 1.5 | 6.7 ± 0.7 | > 0.05 |
|
Ceruloplasmin, mg/l |
274.5 ± 18.5 | 377.2 ± 5.7 | < 0.05 |
|
Note: authenticity of distinctions of indices between the groups was found at p < 0.01. |
|||
The decreasing AOA in the serum and the simultaneous decrease in activity of antioxidant enzymes of SOD and CAT in red blood cells in the patients of this group indicate the attenuation and breakdown of various links of the antioxidant system.
Time course of lipid peroxidation and antioxidant system in patients with MI
Table 5
| Time course of values of lipid peroxidation and antioxidant system in the patients with myocardial infarction at various satges of the disease |
|
Indices |
Terms of myocardial infarctioin (24 hours) | |||
|
1st day n = 142 (1) |
3rd day n = 142 (2) |
15th day n = 141 (3) |
Healthy persons n = 30 |
|
|
MDA, µmol/l |
17.4 ± 2.1 *1-2 | 22.8 ± 2.4 | 20.5 ± 2.3 | 5.4 ± 0.6 |
|
DC, µmol/l |
33.2 ± 2.2 | 37.1 ± 2.6 | 36.3 ± 2.3 | 21.6 ± 1.7 |
|
CP, mg/100 ml |
30.5 ± 2.3 *1-2 | 37.1 ± 2.7 | 36.1 ± 2.2 *1-3 | 29.6 ± 1.1 |
|
CAT, IU/mg Hb |
433.2 ± 11.1 | 433.7 ± 12.0 | 422.7 ± 9.2 | 444.0 ± 11.0 |
|
SOD, c.u./mg Hb |
8.6 ± 0.5 | 8.6 ± 0.6 | 8.3 ± 0.3 | 8.5 ± 0.7 |
| AOA, % | 50.6 ± 3.4 | 44.7 ± 3.1 | 44.2 ± 3.0 | 69.5 ± 2.7 |
|
Antioxidant state factor, c.u. |
0.164 ± 0.01**1-2 | 0.234 ± 0.002 | 0.201 ± 0.002 | 0.031 ± 0.001 |
|
Note:* - authenticity of distinctions of indices at ð < 0.05. |
||||
The data in the table 5 shows the activation of the lipid peroxidation processes and breakdown of antioxidant protection within the first hours after MI. On the third day of the disease the levels of the lipid peroxidation products in blood plasma were characterized by higher values as compared with the corresponding values within the first 24 hours, whereas general AOA of the blood was still decreasing. On the 15th day after development of MI we could observe the unreliable decrease in MDA and DC in blood plasma, whereas the features of the changes in AOS were without any significant time trends in comparison with the 3rd day after MI. Currently, the participation of the processes of free radical lipid oxidation processes in ischemic myocardial injury has been proved [1]. The results of multiple clinical and experimental studies show the activation of the lipid peroxidation processes and breakdown of AOS in patients with MI [18]. Also it has been confirmed by our study.
The comparison of the time courses of the correlation between the parameters of AOS and lipid peroxidation showed the increase in conjugation between the level of intermediate and end products of lipid peroxidation on the 15th day after MI. Moreover, the coefficient of correlation between the plasma level of DC and MDA showed the consistent reliable increase from r = 0.511 (p < 0.05) on the first day to 0.734 (p < 0.01) and 0.745 (p < 0.01) on the 3rd and 15th days after appearance of the disease correspondingly. Also the negative correlation was found between MDA and DC in plasma from one side, and total plasma AOA from other side. The highest values of the correlation relationships between MDA and AOA, DC and AOA in plasma were found on the 3rd day after MI (r = -0.472; p < 0.05 and r = -0.651; p < 0.01 correspondingly). It can testify the intensification of the processes of lipid peroxidation and antioxidant protection within that period of the disease. It is confirmed by the highest plasma levels of MDA and DC on the 3rd day after MI.
According to the opinions by some authors, patients with coronary heart disease show the degree of accumulation of the lipid peroxidation products in the blood which is directly proportional to severity of the disease. The correlation relationship was found between the parameters of lipid peroxidation and AOS from one side and the square of the affected myocardium from other side. The correlation analysis showed the absence of any reliable relationship between the square of the necrotizing myocardium and the parameters of lipid peroxidation and AOS on the days 1 and 3 after MI. The absence of a correlation relationship between these parameters in acute phase of MI, which was found in the present study and previous ones [1], has confirmed the well-known results of the experimental researches of possible activation of the lipid peroxidation processes both in the necrotic region and in adjacent uninjured regions of the myocardium. According to V.Z. Lankin et al. (2000), this process may be responsible for extension of the necrotic region of lesion, because activation of the processes of free radical oxidation in the ischemic region adjacent to necrosis aggravates necrotic degeneration of ischemic cells [10]. Moreover, the reliable negative correlation was found between ceruloplasmin and SOD from one side and the square of the injured myocardium from other side. After summarizing the results one can suppose that the reparative processes in the necrosis region are stimulated by the antioxidants of enzymatic and non-enzymatic origin on 15th day after MI.
CONCLUSION
Some changes in the lipid peroxidation status are observed in development of experimental chronic pancreatitis and in primary chronic pancreatitis in patients with myocardial infarction. Disbalance in pro- and antioxidant system is manifested in view of suppression of plasma antioxidant activity and intensifying the processes of lipid peroxidation.