Measurement of Free Radicals Using Electron Paramagnetic Resonance Spectroscopy During Open Aorto-Iliac Arterial Reconstruction

Background

Aortic cross-clamping during bypass grafting in patients with abdominal aorta aneurysms (AAA) leads to development of ischemia-reperfusion injury. Previous studies have shown that increased production of reactive oxygen species (ROS), such as superoxide anions, hydroxyl radicals, or hydrogen peroxide, plays a major role in cellular damage during post-ischemic reperfusion [1,2]. Free radicals can participate in reduction-oxidation reactions, initiate lipid oxidation, degrade nucleic acids and membrane proteins, and increase endothelial adhesion molecule synthesis. However, free radicals as a result of the aortic cross-clamping during the surgical reconstruction, induce proinflammatory cytokines and chemokines [3]. The synthesis of cytokines during the inflammatory processes generates an increase in concentrations of acute-phase proteins as a response to the tissue destruction [4].

Endogenous antioxidant enzymes – superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and heme oxygenase (HO) – have been shown to prevent skeletal muscle ischemia reperfusion injury by different mechanisms inhibiting ROS activity [5]. When a bypass graft patient develops imbalance between free radicals and antioxidants, it has been shown that it leads to oxidative tissue damage. In patients undergoing AAA bypass grafting, it is possible to reduce oxidative stress and cellular damage by introducing different types of antioxidant treatment such as vitamin E and C, statin, xanthine oxidase, or mannitol. Several reports have described cases of elective AAA patients in whom reperfusion during bypass grafting led to oxygen free radicals production resulting in lipid peroxidation, especially in rupture AAA [6–9]. However, there have been no comparative studies on production and inhibition of ROS in patients with AAA and aortoiliac occlusive disease (AIOD) with critical and moderate ischemia.

Identification of free radicals is commonly investigated by using indirect methods and measurements, such as conjugated dienes, hydroperoxides, and aldehydes [10]. Electron paramagnetic resonance spectroscopy (EPR) spin-trapping has been considered a valuable method because it directly measures free radicals production [11]. EPR is widely used in investigating the structure and dynamics of paramagnetic centers and free radicals that have 1 or more unpaired electrons. The main advantages of EPR spectroscopy are simple preparation of the sample, short time of measurement, and high sensitivity. These advantages are why EPR is becoming widely used in studies of biological samples. This method is based on interaction of an external magnetic field with magnetic moments of unpaired electrons in a sample, which leads to splitting the electron energy levels. An EPR signal is observed when the quantum of the electromagnetic wave energy (hν) incident on the sample is equal to the energy difference between the energy levels, which is described by the resonance condition:

where g is the so-called g-factor, μB is the Bohr magneton, and B0 is the magnetic induction of an external magnetic field. The value of the g-factor can provide information about the kind of paramagnetic center, and the intensity of EPR signals is directly proportional to the free radical concentration in a sample. For this reason, many biological substances have been investigated by this method [12–14].

Therefore, in the present study the technique of electron paramagnetic resonance spectroscopy was used to examine the rate of free radicals generation in relation to the activity of antioxidant enzymes and proinflammatory cytokines production in AAA and AIOD patients treated surgically.

Material and Methods

ETHICAL APPROVAL:

Institutional regulatory board approval 1021/08 16^th October 2008.

STATISTICAL ANALYSIS:

The results were analyzed by a non-parametric Mann-Whitney U test. To compare the median values between the 2 dependent groups at different times before and following surgical treatment, the Wilcoxon matched-pairs signed-ranks test was applied with Bonferroni correction. Spearman’s correlations were performed for correlation analyses. Statistical significance was accepted at the level of P<0.05.

Results

The highest radicals concentration was observed after aortic clamp removal. The measured median values of free radicals by EPR before induction of anesthesia, pre-release of aortic clamp, 5 min after aortic clamp removal, and reperfusion of first leg, 5 min after ipsilateral branch of prosthesis clamp removal, and reperfusion of second leg and 24 h after the operation are summarized in Table 2. Median values of the free radicals revealed a significant elevation after 5 min of the reperfusion of the first leg in surgical treatment of patients with AAA. In both groups of patients, 24 h after surgery, free radicals concentration decreased to a value similar to that before the operation. The values of MDA 5 min after aortic clamp removal and reperfusion of the first leg and also after the reperfusion of the second leg were increased significantly in AAA (Table 3). The changes in free radicals level significantly was correlated with the time of aortic cross-clamping after the reperfusion of the first and second leg in patients with AAA (r=0.7; r=0.47). Typical spectra of AAA samples are presented in Figure 1.

The median values of ox-LDL concentration in AAA patients were significantly decreased 5 min after reperfusion of the first leg (32.99 U/L, range: 14.09–77.12) and 5 min after reperfusion of the second leg (26.75 U/L, range: 11.56–82.12) as well as 24 h after the operation (25.85U/L, range; 14.29–49.70) in comparison to the results obtained before the operation (42.27 U/L, range: 25.53–127.78). In the patients with AIOD, decreased values of ox-LDL concentration after reperfusion of the first leg: 30.13 U/L (12.05–116.19) and second leg: 27.16 U/L (9.92–55.33) and also 24 h after the operation: 27.18 U/L (9.38–57.30) were not significant as compared to the value observed before the operation 37.77 U/L (17.10–101.36).

The concentrations of HO-1, and the activity of GPx and SOD in AAA and AIOD before and during the surgical treatment are summarized in Table 4.

The following concentration medians of CRP, IL-6, and TNF-alfa before the operation were found in the AAA patients: 1.21 mg/l (0.2–3.24), 84.24 pg/ml (9.49–241.31), 3.77 pg/ml (0.44–58.45), respectively, and in the AIOD patients: 2.37 mg/l (1.53–6.84), 71.25 pg/ml (60.89–141.91), 3.13 pg/ml (0.39–24.85), respectively. Twenty-four hours after the surgery, inflammatory markers increased in the patients with AAA: CRP was 14.76 ml/l (0.23–38.55), IL-6 was 141.22 pg/ml (84.3–591.03), and TNF-alfa was 6.82 pg/ml (1.76–80.01). In the patients with AIOD, CRP was 18.44 mg/l (2.56–33.14), IL-6 was 184.1 pg/ml (128.46–448.03), and TNF-alfa was 7.74 pg/ml (1.74–74.74).

Discussion

Reperfusion of ischemic tissue due to cross-clamping of the aorta following AAA surgery induces an inflammatory response and free radicals production [15]. Using EPR and the nitrosobenzene spin trap, we found a significant increase in the AAA group in free radicals production during reperfusion of the first leg 5 min after the aortic clamp removal, but after reconstruction of blood flow to the second leg this elevation of free radicals was insignificant. Detected radicals are probably extracellular. In patient with AOID in which there was lack of ischemic period related to cross-clamping, no significant elevated free radicals level was observed in the reperfusion phase. Twenty-four hours after the operation, free radicals decreased to the level observed before surgery in both groups. In the first minutes of reperfusion and reoxygenation of hypoxic tissue, high formation of oxygen radicals such as superoxide radicals (O–2) hydroxyl radicals (.OH) and hydrogen peroxide (16) is observed. The superoxide radicals in the reperfusion phase react with nitric oxide (NO) to generate peroxynitrite anions, which leads to the peroxidation of lipid and membrane proteins [17]. Our data demonstrated significant increase in MDA concentration during reperfusion in the AAA group in comparison to the AOID group (Table 3). MDA is a final product of lipid peroxidation, derived from polyunsaturated fatty acids, and is used for estimation of free radicals produced by ischemic and reperfused legs [18].

Increased formation of reactive oxygen radicals during reperfusion is associated with different mechanisms of mitochondrial function, DNA damage, activation of apoptosis, destruction of cellular membranes, capillary perfusion failure, interstitial edema, and multiorgan dysfunction syndrome [19–21]. Severity of ischemia and reperfusion cellular injury is dependent on overproduction of free radicals, duration of ischemia, degree of surgical trauma, endogenous antioxidant capacity, and antioxidant therapy.

Our study demonstrated in the AAA group that among endogenous antioxidants HO-1 was significantly decreased 5 min after reperfusion of the first leg and the second leg. The beneficial role of HO-1 is related to protection of tissues against oxidative stress [22]. In addition, GPx and SOD were decreased 5 min after reperfusion of the first leg in patients with AAA, but SOD was also decreased in early reperfusion in patients with AOID [23]. SOD converts superoxide anions to hydrogen peroxide and then hydrogen peroxide is transformed to water and oxygen by glutathione peroxidase and catalase [24]. This activity of the GPx, SOD, and HO-1 levels was associated with an increase in production of free radicals during reperfusion of the first leg. To protect tissue against injury from peripheral ischemia and reperfusion, anesthetic drugs such as propofol and sevoflurane were administered to all the patients. Propofol is chemically similar to α-tocopherol and may decrease MDA level and inhibit lipid peroxidation [25–28]. Other drugs useful in attenuating the production of free radicals (e.g., statins, β-blockers, ACE inhibitors, heparin, and mannitol) were administered according to Table 1 [29–35]. Reperfusion was performed in 2 different periods and separately in both legs, containing a smaller area of ischemic tissue and creating better conditions for antioxidant activity to prevent excessive free radicals generation. There are several studies proving that ischemic post-conditioning decreases free radicals production by limiting glutathione oxidation, as well as reducing the incidence of postoperative complications such as myocardial infarction and renal failure [36–43].

A significant increase in CRP, IL-6, and TNF-α observed 24 h after the surgical treatment of patients with AAA and AIOD may not only cause tissue injury during surgery, but also cause muscle ischemia during temporary aortic clamping and reperfusion. In our study, we observed lack of significant correlation between inflammatory markers and production of free radicals during reperfusion. These markers decreased 6–8 days after the aortic reconstructive surgery in uncomplicated cases (54). The endogenous antioxidant enzymes, as well as antioxidant treatment, as shown in our study, are able to prevent increase of ROS during aortoiliac reconstruction of AIOD patients due to lower production of free radicals in comparison to the AAA group, probably as the result of already established collateral flow. Collaterals maintain sufficient blood supply in AOID (Rutherford 3) patients during aortic cross-clamping.

Conclusions

Using EPR spin-trapping, we demonstrated that in an early phase of reperfusion a temporarily elevated level of free radicals contributes to the reaction against ischemia of tissue, leading to a decrease in antioxidants such as HO-1, GPx, and SOD during surgical treatment of AAA. These elevated free radicals levels at the beginning of reperfusion decrease 24 h after surgery due to various endogenous antioxidants and antioxidant therapies. Surgical management including ischemic post-conditioning before starting reperfusion additionally make it possible to prevent development of irreversible tissue damage, morbidity, complications, and mortality in surgically treated patients.