Molecular detection and charcaterisation of biologically relevant free radicals during surgical ischaemia-reperfusion

  • Mariusz Gutowski

    Student thesis: Doctoral Thesis


    Oxygen is one of the most important molecules in human beings. Our research is focused on how the human body can respond and adapt to the physiological challenge posed by a lack of oxygen. Ascorbic acid (Vitamin C) is one of the most important and considered the most effective water-soluble, chain-breaking antioxidant in human plasma, with the capacity to prevents damage by free radicals. This thesis presents four studies investigating the phenomenon of Reactive Oxygen Species (ROS) generation in the many different surgical conditions in the animal and in the human. Study one investigated the geometry and thermodynamic properties of vitamin C. Calculations were carried out at the restricted and unrestricted B3LYP/6-31+G(d,p), B3LYP/6-311++G(d,p) and B3LYP/EPR-II levels for two conformers (1 and 2) of L-ascorbic acid and their respective oxidation products to monodehydroascorbates of ab-initio methods by Gaussian O3W package. Conformer 1, free radical properties are compared with previously published calculations in the gaseous and aqueous solution states and with experimental EPR values. Calculated molecular structures, EPR (electron paramagnetic resonance spectroscopy), the vibration spectral and energetic properties and all are reported including some proposed changes to previous EPR assignments. Conformer 2 of L-ascorbic acid is predicted to have lower energy than Conformer 1, under the method and basis sets used, by between 11 and 26 kJ mol-1 and is stabilised by internal hydrogen bonding. Relaxed potential energy surface (PES) scans were carried out for two proton transfer processes and relative energies of stable minima and barriers between them determined. Hydrogen transfer is predicted in two systems with favourable spatial arrangements of O–H and O groups for which relaxed potential energy surface scans are reported. Calculated vibrational wavenumber values are provided for selected C=C, C=O, C–H and O–H modes assigned to particular groups and significant calculated EPR hyperfine coupling constants (HCC) values for splitting by H(1) and C(13) for radical species are also reported. These calculations contribute to a better understanding of the complex role of L-ascorbic acid and its various oxidised, neutral, ionic and radical forms in biochemistry and medicine. Study two examined if vitamin C could ameliorate the damaging effects of I-R on myocardium and we postulated that the mechanism of vitamin C protection against iii I-R-induced cell death involved quenching of ROS. In the vitamin C group after 5 min of reperfusion a significant, sudden increase of diastolic pressure in the heart was noted and reached a maximum of 77 mmHg after 12 min of reperfusion and then gradually decreased to 51 mmHg after 60 min of reperfusion period but was quicker than in Control group reaching 37 mmHg by the end of the reperfusion period. The level of A·− (ascorbate free radicals) sudden and massive increased at the time of reperfusion in the Vitamin C group. This increase was associated with poor mechanical function in hearts as indicated by the significantly depressed recovery process. After 30 min of global, now-flow ischaemia and 60min of reperfusion infarct size averaged 33% ± 1 in Control group and 30 % ± 1 in Vitamin C group, respectively, (P<0.05). There is strong evidence that oxygen centered radicals contribute to postischaemic dysfunction after global ischaemia. Our data unquestionably suggest that the large production of A·− was associated with a greater depression in myocardial contractile function, therefore could represent a marker of oxidative stress during I-R and could be related to the functional impairment during reperfusion. In summary, we have used the animal models of isolated heart perfusion to provide evidence that vitamin C did not reduce the infarct size, however “tendency” towards a decrease (↓) in infarct size with ascorbate and it protects from oxidative damage during global I-R as manifested by decreased concentrations of A·− and enhance recovery of mechanical function such as diastolic pressure and LVDP in postischaemic working rat hearts. Study three was designed to test the hypothesis that the physiological trauma associated with venous cannulation may artefactually stimulate systemic free radical formation in the acute phase that if not accounted for may under-estimate the oxidative stress response to exercise. The relationship between the time of venepuncture and the level of free radical generation during normoxic conditions was further investigated. The venous cannulation in Phase I, increased plasma A·− by 347 ± 173 AU/√G, P <0.05 after 2min of venepuncture with further increases observed after 5min and 10min of venous cannulation, respectively (403 ± 178 AU/√G; 462 ± 93 AU/√G, P < 0.05) vs baseline point time. After this time the level of A·− slightly blunted as to achieve a similar level to baseline point control after 30 minutes. In phase II the exerciseinduced increase in A·− was subsequently shown to be 48% greater (30min as opposed to the 2min post-cannulation resting baseline)(1754 ± 361 vs. 1979 ± 375 AU, P <0.05). Our findings demonstrate and confirm that venous cannulation per se stimulates iv the systemic formation of free radicals as an acute phase response which peaks at 10min and require approximately 15min to normalise. This has important interpretive implications for future studies that employ catheterisation. The final Study examined if the combination of exercise and inspiratory hypoxia would further compound regional tissue de-oxygenation that is frequently encountered during the ischaemic phase of surgery and thus, by consequence increase oxidative stress. The aim of the study was to further understand a potential relationship between oxidative stress and alterations in muscle oxygenation. Clear significant increases in the plasma concentration of A·− were detected in the peripheral blood of patients (normoxia(baseline) vs 6 data points of reperfusion after 5min of global ischaemic condition, P<0.05),(baseline vs immediate after ischaemia; 2337±525 vs 2633±508, AU, respectively). During global ischaemia the regional muscle oxygenation significantly decreased (↓∆O2Hb-oxyhaemoglobin), ↑∆HHb- deoxyhaemoglobin ), although increased regional blood volume (↑∆tHb- total haemoglobin). From the end of global ischaemia to 10 min after the regional muscle oxygenation progressively back to the start data point (↓∆HHb, ↑∆O2Hb). This study demonstrates for the first time that the I-R has got a big influence on the muscle oxygenation to increased ROS and the return of values towards baseline period in reperfusion stage appears to coincide with increased oxidative stress. Moreover, the present study has also demonstrated increased A·− level as early as the ischaemic phase of experiment independent of perioperative changes in the partial pressure of oxygen (pO2), elucidate a potentially important role for oxidative stress in provoking an appropriate vasodilation (NO-bioavailability) during the I-R period. This work demonstrates that; - Ascorbate is an antioxidant that can scavenge tissue and blood borne free radical, is essential in controlled amounts and is capable of initiating protective adaptation in the face of oxidative stress for the maintenance of physiological homeostasis. - Reperfusion is always associated with a sudden and massive release of ascorbate free radicals, with a maximal liberation within the first minutes of reperfusion. Vitamin C tended to reduce infarct size and protects from oxidative damage during global ischaemia and reperfusion. - The venous cannulation alone is enough per se stimulates the systemic formation of free radicals as a acute phase response. If this baseline artefact is not taken into account, the true magnitude of the exercise-induced oxidative stress response will be under-estimated. - The I-R has got a major influence on the muscle oxygenation to increased ROS and the return of values towards baseline period in reperfusion stage appears to coincide with increased oxidative stress. Using the state-of-the-art molecular techniques that include Electron Paramagnetic Spectroscopy (EPR) for the direct detection of free radicals and Near Infrared Spectroscopy (NIRS) for the direct detection of muscle oxygenation these studies have attempted to translate the basic mechanisms associated with free radical formation during I-R and have provided unique insight into the basic mechanisms responsible for the oxidative stress with the ultimate objective of developing novel antioxidant interventions that can provide effective prophylaxis.
    Date of AwardApr 2011
    Original languageEnglish
    SupervisorDamian Bailey (Supervisor), William George (Supervisor) & William Ford (Supervisor)


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