Evidence Of Free Radical Production Research Articles

Oxidative Stress and Serum Selenium in HIV Patients on Different Antiretroviral Regimen

Background: Two major Highly Active Antiretroviral Therapy naming as HAART-1(zidovudine, lamivudine and nevirapine) and HAART-2 (zidovudine, lamivudine and efavirenz) are considered for HIV positive patients. HIV as well as antiretroviral drugs can cause free radical formation with or without compensatory increase in serum selenium. This study was aimed at knowing serum levels of malonaldehyde (evidence of free radical production) and selenium. Method: A total of 240 persons were surveyed, comprising subjects and controls. The respondents were in four groups each comprising 60 persons. The four groups are: HIV positive patients not yet on HAART termed Prehaart patients, HIV positive patients on HAART-1, HIV positive patients on HAART-2 and HIV negative patients (control). Serum levels of malonaldehyde (MDA) and selenium were assayed in each of study groups. Result: CD4/mm 3 was significantly lower in subject groups when compared with control (p<0.05). MDA (µmol/l) was found to be higher in all subject groups but significant elevation was only found in HAART-2 group (p<0.05). Serum Se (µg/dl) levels were found to be significantly lower in all the groups when compared with control (p<0.05). Conclusion: Production of free radicals was worse in patients on HAART-2. Decreased serum selenium levels in all groups may suggest reason for oxidative stress found in HIV positive patients on antiretroviral therapy. Abbreviation: HAART-1(zidovudine, lamivudine and nevirapine), HAART-2(zidovudine, lamivudine and efavirenz), MDA (Malonaldehyde),Se (Selenium)

Role of Lipid-Derived Free Radical in Bleomycin-Induced Lung Injury in Mice: Availability for ESR Spin Trap Method with Organic Phase Extraction

Bleomycin is well known as causative molecule for acute lung injury and interstitial pneumonia. The free radical production from bleomycin is thought to play an important role in the pathogenesis of acute lung injury and interstitial pneumonia. However, there was no direct evidence of free radical production in this model. Therefore, we examined in vivo radical production by mice treated with a bleomycin using electron spin resonance with the spin trap, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. Six hours after instillation of bleomycin, the lung exposed to bleomycin gave a lipid-derived free radical adduct, which would support evidence for in vitro lipid peroxidation resulting from bleomycin administration. In the treatment of deferoxamine, chelating agent for iron and other metals, to reduce the bleomycin induced free radical production, parallel to decrease the lipid-derived free radical production by deferoxamine, pathophysiological findings of lung injuries were improved by deferoxamine. In conclusion, this is a first paper of in vivo direct evidence of production of free radical from bleomycin-induced lung injury. It is suggested that this method may be used in many kinds of lung disease models, which have potentials of free radical production to cause lung damage.

In vivo activation of N-methyl-D-aspartate receptors in the rat hippocampus increases prostaglandin E(2) extracellular levels and triggers lipid peroxidation through cyclooxygenase-mediated mechanisms.

Cyclooxygenases (COX) are a family of enzymes involved in the biosynthesis of prostaglandin (PG) and thromboxanes. The inducible enzyme cyclooxygenase-2 (COX-2) is the major isoform found in normal brain, where it is constitutively expressed in neurons and is further up-regulated during several pathological events, including seizures and ischaemia. Emerging evidence suggests that COX-2 is implicated in excitotoxic neurodegenerative phenomena. It remains unclear whether PGs or other products associated to COX activity take part in these processes. Indeed, it has been suggested that reactive oxygen species, produced by COX, could mediate neuronal damage. In order to obtain direct evidence of free radical production during COX activity, we undertook an in vivo microdialysis study to monitor the levels of PGE(2) and 8-epi-PGF(2alpha) following infusion of N-methyl-D-aspartate (NMDA). A 20-min application of 1 mm NMDA caused an immediate, MK-801-sensitive increase of both PGE(2) and 8-epi-PGF(2alpha) basal levels. These effects were largely prevented by the specific cytosolic phospholipase A(2) (cPLA(2) ) inhibitor arachidonyl trifluoromethyl ketone (ATK), by non- selective COX inhibitors indomethacin and flurbiprofen or by the COX-2 selective inhibitor NS-398, suggesting that the NMDA-evoked prostaglandin synthesis and free radical-mediated lipid peroxidation are largely dependent on COX-2 activity. As several lines of evidence suggest that prostaglandins may be potentially neuroprotective, our findings support the hypothesis that free radicals, rather than prostaglandins, mediate the toxicity associated to COX-2 activity.

IN VIVO ASSESSMENT OF FREE RADICAL ACTIVITY DURING SHOCK WAVE LITHOTRIPSY USING A MICRODIALYSIS SYSTEM: THE RENOPROTECTIVE ACTION OF ALLOPURINOL

Shock wave lithotripsy is believed to cause renal damage directly through cellular injury from high energy shock waves and indirectly through vascular injury and resultant ischemia, which gives rise to oxygen free radical compounds. The transient and volatile nature of free radicals and derived products makes their detection difficult. Moreover, certain medications may provide a protective effect against shock wave lithotripsy induced renal parenchymal injury. We introduced an innovative microdialysis system for in vivo sampling of interstitial fluids that can be analyzed for free radical mediated lipid peroxidation products after shock wave lithotripsy treatment in the swine model. In addition, this system was used to test the antioxidant or renoprotective action of allopurinol. Ten juvenile swine were assigned to a nonmedicated control group that underwent shock wave lithotripsy or to a group that was premedicated with allopurinol before shock wave lithotripsy. Each group of animals underwent shock wave lithotripsy to the lower pole of the right kidney and received a total of 10,000 shock waves. Dialysate fluid was collected at 1,000-shock wave increments via probes surgically implanted into the lower pole of the right and left kidneys before lithotripsy. Samples were immediately preserved in liquid nitrogen and subsequently analyzed for the presence and concentration of conjugated diene levels, a measure of lipid peroxidation. Five additional juvenile swine were assigned to a sham treated group that did not undergo shock wave lithotripsy. Dialysate fluid was collected from the lower pole of the right and left kidneys to establish baseline or pre-lithotripsy levels of conjugated dienes. After shock wave lithotripsy conjugated diene levels increased almost 100-fold over that in the right kidneys of the nonmedicated control group. The difference was statistically significant compared to levels in the contralateral untreated kidneys (p <0.01). Right kidneys in the group premedicated with allopurinol did not demonstrate an increase in conjugated diene levels during shock wave lithotripsy. The results of this study confirm shock wave lithotripsy induced free radical activity as well the antioxidant and protective nature of allopurinol. The newly described microdialysis system enables real-time sampling of interstitial fluids during shock wave lithotripsy. It represents a unique method for assessing free radical formation and evaluating the protective effects of additional antioxidant medications.

Hyperbaric oxygen therapy and free radical production: an experimental study in doxorubicin (Adriamycin) extravasation injuries.

The role of hyperbaric oxygen (HBO) therapy in free radical-mediated tissue injury is not clear. HBO has been shown to enhance the antioxidative defense mechanisms in some animal studies, but HBO has also been reported to increase the production of oxygen free radicals. To investigate this controversy, we studied the effect of HBO in a doxorubicin (Adriamycin) extravasation model, because the cytotoxic activity of doxorubicin is partly related to its quinone structure, which leads to the formation of cytotoxic oxygen intermediates. Fifty-four Sprague-Dawley rats underwent injection of 0.3 ml doxorubicin solution (2 mg per milliliter) intradermally on both flanks as described by Rudolph and colleagues. Group I (N = 28) received HBO treatment (2 hours at 2 ATA) for 3 days prior to injection and 7 days postinjection. Group II (N = 26) received no HBO treatment. At 2, 3, and 5 weeks, the size of the ulcers and the surrounding area of alopecia in group I (+HBO) were significantly larger than in group II (-HBO): 112.2 mm2 vs. 42.8 mm2 (p < 0.01) and 1,132.2 mm2 vs. 364.8 mm2 (p < 0.005). Biochemical analysis of the biopsied skin ulcers, to measure the parameters of oxygen free radical production, indicated (similar) low levels of xanthine oxidase for both groups. However, significantly elevated levels of malonyldialdehyde (MDA), indirect evidence of free radical production, was observed in group I (+HBO) in comparison with group II (-HBO): 36.58 vs. 5.84 ng per minute per milligram protein (p < 0.001), which might indicate free radical-induced cellular injury. It is concluded that in this animal study the cytotoxicity of doxorubicin is potentiated by HBO therapy. The elevated levels of MDA suggest a direct additive cytotoxic effect by increased membrane lipid peroxidation. HBO therapy, therefore, might be deleterious in the early (preulcer) stage of doxorubicin extravasation.

Plasma antioxidant consumption associated with ischemia/reperfusion during carotid endarterectomy.

Thirteen patients undergoing carotid endarterectomy were studied to correlate changes in jugular bulb venous oxygen saturation (SjO2) with indices of free radical production during cerebral ischemia/reperfusion. Levels of oxidant species were also monitored in arterial samples to determine any change across the cerebral circulation. Blood was sampled from a venous catheter inserted in the ipsilateral jugular bulb and from an arterial catheter. Co-oximetry measurements were made to determine jugular bulb venous oxygenation saturation. To monitor changes in oxidant stress, a colorimetric assay was used to determine plasma antioxidant potential, and electron paramagnetic resonance spectroscopy was used to quantify free radical-spin trap adducts formed in a blood sample treated with the spin trap alpha-tert-butyl phenyl nitrone (PBN). SjO2 decreased significantly from 68 +/- 11% to 61 +/- 10% (P < .05) during clamping of the internal carotid artery and returned to baseline (65 +/- 11%) when the carotid clamp was removed. Jugular venous plasma antioxidant potential decreased significantly from 32.76 +/- 5.42% inhibition to 28.02 +/- 6.77% inhibition (P < .05). There was no concomitant change in arterial plasma antioxidant potential values, indicating a decrease in antioxidant capacity across the cerebral circulation. However, analysis of spin trap-free radical adducts did not provide conclusive evidence for free radical production. These results provide supportive evidence for oxidant production during cerebral ischemia/reperfusion in a clinical setting.

Evidence for free radical generation after primary percutaneous transluminal coronary angioplasty recanalization in acute myocardial infarction

In animal models, oxygen-derived free radicals have been found to be important mediators of reperfusion injury to ischemic but viable myocardium. However, in humans, there is no direct evidence of free radical production after the restoration of coronary artery patency in acute myocardial infarction. The purpose of this study was to quantitate and assess the time course of free radical production in coronary venous outflow in patients with acute myocardial infarction undergoing successful recanalization of the infarct-related artery by primary percutaneous transluminal coronary angioplasty (PTCA). Primary PTCA was performed in 17 patients with acute myocardial infarction of <6 hours duration. Direct free radical production was assessed by coronary venous effluent blood sampling before PTCA and at timed intervals up to 24 hours (or 48 hours in 6 patients) after recanalization. All samples were added to the spin trapping agent α-phenyl N-tert butyl nitrone and analyzed by electron paramagnetic resonance spectroscopy. Vessel patency resulted in a sharp increase in free radical signal. Relative to the level before PTCA, the changes reached statistical significance after only 15 minutes (p <0.05). Peak signals were observed between 1 1 2 and 3 1 2 hours (p < 0.001), then declined up to 5 hours. A second increase in signal level was detected between 18 and 24 hours despite no angiographic evidence of reocclusion. A gradual decline was observed after 24 hours. These findings provide the first direct and quantitative evidence of free radical production in the immediate postrecanalization phase after thrombotic occlusion of a major coronary artery in humans.

Direct evidence of free radical production after ischaemia and reperfusion and protective effect of desferrioxamine: ESR and vitamin E studies

After surgical renal revascularisation, warm renal ischaemia due to renal artery cross-clamping contributes to postoperative renal dysfunction. After reperfusion, free radicals are thought to be a significant cause of injury. Nevertheless, indisputable proof of free radical production is scarce, partly because of their transient nature. In this study, electron paramagnetic resonance and vitamin E levels were used to demonstrate the free radical production after renal ischaemia and reperfusion. Rabbit kidneys were submitted either to 15 or 60 minutes of ischaemia followed by reperfusion. A spin trap agent (alpha-phenyl-N-tert-butyl nitrone (PBN), 20mg/ml, 1 ml/min) was infused during reperfusion directly into the left renal artery via an aortic catheter before declamping. Blood samples were selectively drawn from the left renal vein for ESR analysis (Varian spectrometer E109) of lipidic residues extracted from blood samples. The vitamin E content of the left renal cortex was determined by HPLC procedure. The right renal cortex was used as a control for the vitamin E values. In the venous effluent, ESR analysis revealed the formation of a spectrum consisting of a triplet of asymmetric doublets. This signal resulted from the spin trapping by PBN of a mixture of both oxygen- and carbon- centred lipidic radicals. The amplitude of the signal which is proportional to the amount of free radicals was significantly higher after 60 minutes ischaemia than after 15 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Free radicals and septic shock in primates: The role of tumor necrosis factor

The role of free radicals in septic-shock-associated tissue injury and the mechanisms underlying the generation of free radicals in sepsis was investigated in a primate model using electron spin-trapping techniques paired with physiological measurements. Baboons were administered the spin trap resonance (ESR) spectroscopy and spin-trapping techniques paired with physiological measurements. Baboons were administered the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) during infusions of live Escherichia coli(E. coli) with or without challenge with tumor necrosis factor (TNF). ESR spectra suggesting the trapping of carbon-centered and oxygen-centered radicals were detected in liver lipid extracts of E. coli infused animals which exhibited patholophysiological changes indicative of sepsis. In animals demonstrating a toxic response to E. coli, TNF challenge appeared to intensify the ESR signal observed. The data provide evidence of free radical production during sepsis and suggest a role for TNF in the production of these radicals.

Free radicals and myocardial reperfusion injury.

Ischaemic myocardial tissue will, inevitably, necrose if blood flow is not restored. Whilst reperfusion is always beneficial in terms of potential recovery of heart muscle, reperfusion in itself is believed to bring about cellular injury. While the causes of this 'reperfusion injury' are apparently multifactorial, there is now an increasing body of evidence to suggest that oxygen free radicals play a major role in the pathogenesis of reperfusion injury. The initial evidence for this hypothesis was indirect, based on the ability of free radical scavengers to limit myocardial injury in animal models. More recent work has utilised the highly specific technique of electron spin resonance (ESR) spectroscopy and ESR spin trapping to detect the free radical species. The evidence for free radical production on myocardial reperfusion will be presented along with details of human studies. The potential for a therapeutic intervention will also be briefly discussed.

Myocardial vitamin E is consumed during cardiopulmonary bypass: indirect evidence of free radical generation in human ischemic heart

Although a role for free radicals in myocardial damage during cardiopulmonary bypass for open heart surgery has been postulated, direct evidence of free radical production as well as consumption of tissue antioxidants such as vitamin E is still lacking. Twenty patients (age 26–66 yr, mean 48) undergoing elective open heart surgery with moderate hypothermia, and cold crystalloid cardioplegia, were studied. Cardiopulmonary bypass time was 61.4 ± 31.2 min. The specimens of atrial tissue collection before and after cardiopulmonary bypass, were immediately frozen in liquid nitrogen. Mean vitamin E atrial content, measured by reverse phase HPLC, was 355 ± 249 pmol/mg of dry weight basally, 135 ± 85 pmol/mg ( p < 0.05) at the end of the ischemic period and 405 ± 288 pmol/mg after the reperfusion period ( p < 0.01). Microscopic examination of right atrial biopsies ruled out differences in fibrosis or cellular damage as the cause of vitamin E changes. Although a great basal variability in atrial vitamin E content was observed, which was independent of age, sex and clinical status, a reproducible and substantial decrease in atrial vitamin E content after cardiopulmonary bypass occurred (mean reduction 45 ± 17% and 55 ± 22%, respectively, after ischemia and after reperfusion). This was directly related to the aorta cross-clamping duration and partially to the minimum temperature achieved. In conclusion, apart from the great variability observed in basal vitamin E tissue content, vitamin E was always reduced during cardiopulmonary bypass, suggesting an oxidative stress on the myocardium during open heart surgery.

Could treatment with scavengers of oxygen free radicals minimize complications in cardiac surgery?

Cardiac surgical complications may be caused by a plethora of events. Oxygen free radicals may participate in the pathophysiology of cardiopulmonary bypass and myocardial ischaemia-reperfusion injury. The evidence of free radical production during bypass is limited, and studies of the effect of free radical scavengers even more limited. Experimental work points towards a role of oxygen free radicals in reperfusion damage of the ischaemic myocardium, but clinical data are lacking. Hitherto the clinical availability of free radical scavengers/antioxidants has been scarse. At present more such drugs are becoming clinically applicable, and trials on their effect are highly warranted, in particular as an adjuvant in myocardial protection.

Evidence for free radical production by ultrasonic cavitation in biological media

The statement is often made that free radicals will be produced during the violent collapse of transient cavities in liquids, that may result from the passage of ultrasonic waves in the low megahertz frequency range. Evidence for such free radicals is mostly indirect. We have insonated some argon-saturated aqueous solutions of biological molecules (trypsin, Dulbecco's Minimum Essential Medium, fetal bovine serum, and human blood plasma) and whole human blood, all containing a spin trapping compound, at 1 MHz, continuous wave, 6.6 W/cm 2 (SPTA) for 15 min at 4–8°C. Electron spin resonance spectra of the samples were recorded 10 and 30 min. after cessation of insonation. The spectra included those of spin adducts formed with ·OH and ·H radicals, which were absent in the cases of the sham treated controls. Thus, we have demonstrated the formation of the highly reactive ·OH and ·H radicals in the insonated media, even in the presence of natural radical scavengers in the mammalian derived products.

Evidence for Free-Radical Production in Photosynthesizing Systems

Evidence for Free-Radical Production in Photosynthesizing Systems

Evidence for Free-Radical Production in Living Cells Exposed to Ionizing Radiation

Applications of microwave spectroscopy as an index of the presence of free radicals in specimens of biological materials are discussed. The importance is stressed of this analytical tool in the search for mechanisms of the biologic action of radiation. Qualitative microwave spectroscopy is concerned with determining the type of free radical formed by irradiation and usually requires a detailed analysis of hyperfine structure. Many interesting results have been obtained by this method in investigating irradiated biochemicals such as amino acids, proteins, and nucleic acids. Quantitative microwave spectroscopy yields information on the number or concentration of free radicals as a function of radiation dose and dose rate, of time after irradiation, and of external conditions such as temperature and water content of the specimen or type and pressure of the surrounding gas. This method was applied to biochemicals and also to living cells exposed to ionizing radiation. Details of the experimental technique are summarized. Results are presented from studies on irradiated glycine, irradiated barley embryos, and irradiated seeds with various water contents. 15 references. (C.H.)