Electron Spin Resonance Studies on the Degradation of Hydroperoxides by Rat Liver Cytosol

Incubation of ¹BuOOH (in the concentration range 200μM to 20mM) with rat liver post-microsomal supernatant in the presence of the spin trap DMPO gives three radical species, which can be observed by electron spin resonance spectroscopy. The first of these is the ascorbyl radical (which decreases in concentration with time), the other two are identified as spin adducts of alkoxyl and carbon-centred radicals; these latter species increase in concentration with time. Addition of NADH, but not NADPH, led to an increase in concentration of the alkoxyl and carbon-centred radical adducts and a decrease in the concentration of the ascorbyl radical. Results obtained in the presence of iron chelators and other ligands suggest that the generating system is an NADH-dependent enzyme that reduces ¹BuOOH by one-electron to give initially the ¹BUO radical. Results from experiments carried out on dialysed cytosol samples lend support to this conclusion.     

Electron Spin Resonance Studies on the Degradation of Hydroperoxides by Rat Liver Cytosol

Incubation of ¹BuOOH (in the concentration range 200μM to 20mM) with rat liver post-microsomal supernatant in the presence of the spin trap DMPO gives three radical species, which can be observed by electron spin resonance spectroscopy. The first of these is the ascorbyl radical (which decreases in concentration with time), the other two are identified as spin adducts of alkoxyl and carbon-centred radicals; these latter species increase in concentration with time. Addition of NADH, but not NADPH, led to an increase in concentration of the alkoxyl and carbon-centred radical adducts and a decrease in the concentration of the ascorbyl radical. Results obtained in the presence of iron chelators and other ligands suggest that the generating system is an NADH-dependent enzyme that reduces ¹BuOOH by one-electron to give initially the ¹BUO radical. Results from experiments carried out on dialysed cytosol samples lend support to this conclusion.     

Direct Detection of Ascorbyl Radical in Experimental Brain Injury: Microdialysis and an Electron Spin Resonance Spectroscopic Study

Abstract: To examine the role played by free radicals in brain injury, we performed experiments to detect radicals in the frontal cortex of rats, using electron spin resonance (ESR) and microdialysis. A dialysis probe was inserted into the frontal cortex, and spin adducts in perfusates were immediately detected by ESR. We obtained a relatively stable doublet signal, with parameters of g = 2.0057 and aH = 0.17 mT. This signal corresponded with that of the ascorbyl radical. Ascorbyl radical in the perfusate collected from the frontal cortex was augmented by microinjection of H₂O₂ and FeCl₂ adjacent to the dialysis probe. When the rats were challenged with cold-induced brain injury, ascorbyl radical and lactate dehydrogenase (LDH) level in the perfusate increased significantly. Pretreatment with superoxide dismutase and catalase attenuated the increase in ascorbyl radical and LDH level induced by the cold injury. Infusion of FeCl₂ dissolved in perfusate caused a pronounced increase in ascorbyl radical and LDH level after the cold injury. We conclude that the direct detection of free radical formation further supports the hypothesis that free radicals play an important role in traumatic brain injury. Our findings also indicate that combined microdialysis with ESR spectroscopy is a useful in vivo method for monitoring free radical production in the brain.     

Regulation of the Manganese Superoxide Dismutase and Inducible Nitric Oxide Synthase Gene in Rat Neuronal and Glial Cells

Abstract: Bidirectional communication occurs between neuroendocrine and immune systems through the action of various cytokines. Responses to various inflammatory mediators include increases in intracellular reactive oxygen species (ROS), notably, superoxide anion (O₂⁻) and nitric oxide (NO^•). Neurotoxicity mediated by NO^• may result from the reaction of NO^• with O₂, leading to formation of peroxynitrite (ONOO⁻). ROS are highly toxic, potentially contributing to extensive neuronal damage. We, therefore, evaluated the effects of a variety of inflammatory mediators on the regulation of mRNA levels for manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in primary cultures of rat neuronal and glial cells. To determine age-dependent variation of mRNA expression, we used glial cells derived from newborn, 3-, 21-, and 95-day-old rat brains. Interleukin-1β, interferon-γ (IFN-γ), bacterial lipopolysaccharide (LPS), and tumor necrosis factor-α showed significant induction of MnSOD in both glial and neuronal cells. However, only LPS and IFN-γ increased iNOS mRNA. These data demonstrate that these two genes are similarly regulated in two cells of the nervous system, further suggesting that the oxidative state of a cell may dictate a neurotoxic or neuroprotective outcome.     

Superoxide anion, the main species of ROS in the development of ARDS induced by oleic acid

It is believed that reactive oxygen species (ROS) play a very important role in the pathogenesis of acute respiratory distress syndrome (ARDS), but the mechanism has not been so clear, owing to the absence of direct measurable (experimental) data. In majority of the medical studies on free radicals, the analysis of ROS has generally been done by the way of measuring their secondary and breakdown products. In our study, we used electron spin resonance (ESR), a sensitive and accurate technique to detect ROS directly and also used some other sensitive techniques including ultra-weak luminescence and chemical luminescence to identify the species and relative amount of ROS. Furthermore, superoxide dismutase (SOD) was pre-administrated in ARDS rats to verify the results from the above studies and explore the possibility of the clinical application of SOD in ARDS. The spectra of ESR showed that the concentration of ROS increased at 10 min and reached a summit at 30 min after injection of oleic acid (OA), then dropped gradually. The scavenging effects of different scavenging agents on ROS by the analysis of ultra-weak luminescence proved that superoxide anion was the main species of ROS in the development of OA-induced ARDS. Moreover, the results of quantified measure of superoxide anion by chemical luminescence also showed the accordant tendency exhibited in ESR measurement. The pre-treatment of SOD might distinctly inhibit the production of superoxide anion, obviously improve the blood gas status, lung wet/dry ratio and lung/body ratio in ARDS rats. It is suggested that ROS may play a key role in the initiation phase of ARDS, while superoxide anion may be a leading actor in this process and SOD could effectively protect rats from ARDS. These results may provide helpful information for the treatment and prevention of ARDS.     

Superoxide Anion,the Main Species of ROS in the Development of ARDS Induced by Oleic Acid

It is believed that reactive oxygen species (ROS) play a very important role in the pathogenesis of acute respiratory distress syndrome (ARDS), but the mechanism has not been so clear, owing to the absence of direct measurable (experimental) data. In majority of the medical studies on free radicals, the analysis of ROS has generally been done by the way of measuring their secondary and breakdown products. In our study, we used electron spin resonance (ESR), a sensitive and accurate technique to detect ROS directly and also used some other sensitive techniques including ultra-weak luminescence and chemical luminescence to identify the species and relative amount of ROS. Furthermore, superoxide dismutase (SOD) was pre-administrated in ARDS rats to verify the results from the above studies and explore the possibility of the clinical application of SOD in ARDS. The spectra of ESR showed that the concentration of ROS increased at 10?min and reached a summit at 30?min after injection of oleic acid (OA), then dropped gradually. The scavenging effects of different scavenging agents on ROS by the analysis of ultra-weak luminescence proved that superoxide anion was the main species of ROS in the development of OA-induced ARDS. Moreover, the results of quantified measure of superoxide anion by chemical luminescence also showed the accordant tendency exhibited in ESR measurement. The pre-treatment of SOD might distinctly inhibit the production of superoxide anion, obviously improve the blood gas status, lung wet/dry ratio and lung/body ratio in ARDS rats. It is suggested that ROS may play a key role in the initiation phase of ARDS, while superoxide anion may be a leading actor in this process and SOD could effectively protect rats from ARDS. These results may provide helpful information for the treatment and prevention of ARDS.     

Identification and Determination of 1-Methylimidazole-4-Acetic Acid in Human Cerebrospinal Fluid by Gas Chromatography-Mass Spectrometry

Abstract: A method for the quantification of 1-methylimidazole-4-acetic acid in human CSF was developed. Methylimidazole-acetic acid was identified and quantitated in CSF. The method involves concentration of the compound on a cation exchanger, extraction of the methyl ester with ethyl acetate, and preparation of a heptafluorobutyryl derivate of the methyl ester, which is finally purified by chromatography on silica gel and quantitated by gas chromatography-mass spectrometry with the deuterated analogue as internal standard. The coefficient of variation at 1 ng/ml was 13%. The limit of sensitivity was about 0.2 ng/ml. The concentration of methylimidazole-acetic acid in lumbar CSF from healthy volunteers was below 1 ng/ml. Ventricular CSF contained higher concentrations than lumbar fluid. The existence of a rostrocaudal concentration gradient was established. There was a correlation between the concentration of methylimidazole-acetic acid and tele-methylhistamine in CSF. The concentration of methylimidazole-acetic acid in lumbar CSF from schizophrenic patients, patients with subarachnoidal haemorrhage, or patients with rheumatic disease was in the range of that in healthy volunteers.     

Presence and Measurement of Imidazoleacetic Acid, a γ-Aminobutyric Acid Agonist, in Rat Brain and Human Cerebrospinal Fluid

Imidazoleacetic acid (IAA) was unequivocally demonstrated in rat brain, human CSF, and human plasma by a gas chromatographic-mass spectrometric method that can reliably quantify as little as 8 pmol, i.e., 1 ng. Owing to tautomerism of the imidazole ring, IAA and [15N, 15N]IAA, the internal standard, each formed two chromatographically distinct isomers after derivatization of the ring nitrogens with either ethyl chloroformate or methyl chloroformate. The isomers of n-butyl(N-ethoxycarbonyl)imidazole acetate and n-butyl(N-methoxycarbonyl)imidazole acetate were identified by analysis with methane chemical ionization and electron impact ionization of molecular and fragment ions. The levels (mean +/- SEM) of free IAA were 140 +/- 14 pmol/g and 2.7 +/- 0.2 pmol/ml in brains of untreated rats and human lumbar CSF, respectively. Mean levels of IAA in brains of anesthetized rats, perfused free of blood, did not differ significantly from mean levels of anesthetized, nonperfused controls or from untreated rats. The source or sources of IAA in brain and CSF are unknown. Because IAA is a potent agonist at gamma-aminobutyrate receptors, it merits examination as a regulator in brain.     

Superoxide Dismutase Catalyzes Nitration of Tyrosines by Peroxynitrite in the Rod and Head Domains of Neurofilament-L

Abstract: Superoxide dismutase (SOD) catalyzes the nitration of specific tyrosine residues in proteins by peroxynitrite (ONOO⁻), which may be the damaging gain-of-function resulting from mutations to SOD associated with familial amyotrophic lateral sclerosis (ALS). We found that disassembled neurofilament-L (light subunit) was more susceptible to tyrosine nitration catalyzed by SOD in vitro. Neurofilament-L was selectively nitrated compared with the majority of other proteins present in brain homogenates. Assembled neurofilament-L was more resistant to nitration, suggesting that the susceptible tyrosine residues were protected by intersubunit contacts in assembled neurofilaments. Electrospray mass spectrometry of trypsin-digested neurofilament-L showed that tyrosine 17 in the head region and tyrosines 138, 177, and 265 in α-helical coil regions of the rod domain of neurofilament-L were particularly susceptible to SOD-catalyzed nitration. Nitrated neurofilament-L inhibited the assembly of unmodified neurofilament subunits, suggesting that the affected tyrosines are located in regions important for intersubunit contacts. Neurofilaments are major structural proteins expressed in motor neurons and known to be important for their survival in vivo. We suggest that SOD-catalyzed nitration of neurofilament-L may have a significant role in the pathogenesis of ALS.     

Functional Impairment in Protein Kinase C by RACK1 (Receptor for Activated C Kinase 1) Deficiency in Aged Rat Brain Cortex

Abstract: Several laboratories have reported a lack of protein kinase C (PKC) activation in response to various stimuli in the brain of aged rats. It has been suggested that changes in lipid membrane composition could be related to this functional deficit. However, recent evidence has indicated that the translocation of PKC to the different subcellular compartments is controlled by protein-protein interactions. Recently, a class of proteins, termed receptors for activated C kinase (RACKs), have been described that bind PKC. The present study was conducted to determine whether alterations in RACK1, the best-characterized member of RACKs, were associated with changes in translocation and expression of PKC. Quantitative immunoblotting revealed that RACK1 content was decreased by ∼50% in aged rat brain cortex, compared with that in adult and middle-aged animals. The levels of calcium-independent PKCδ and ε, interacting with RACK1, and related calcium-independent PKC activity were not modified by the aging process. By comparison, phorbol ester-stimulated translocation of this activity and of PKCδ and ε immunoreactivity was absent in cortex from aged animals, as well as the translocation of the calcium-dependent PKCβ, also known to interact with RACK1. These results indicate that a deficit in RACK1 may contribute to the functional impairment in PKC activation observed in aged rat brain.     

Free Radicals in Rabbit Spinal Cord Ischemia: Electron Spin Resonance Spectroscopy and Correlation with SOD Activity

1. In nonanesthetized rabbits temporal occlusion of the abdominal aorta was used to induce oxidative stress in the lower part of the body including distal segments of the spinal cord.2. Spinal cord samples were taken from the animals exposed to 25-min aortic occlusion (AO ) or to occlusion followed by 1- or 2-hr reperfusion (AO/R1 or AO/R2, respectively) or from sham-operated animals (C). The presence of free radicals (FR) in the spinal cord samples frozen in liquid N₂ was assessed by ESR spectroscopy without spin trapping. Moreover, superoxide dismutase (SOD) activity and conjugated diene (CD) levels were measured in the samples.3. In the AO group FR were detected in the spinal cord regions close to the occlusion (lower thoracic and distal segments) along with a decrease in SOD activity. The calculated g value (g = 2.0291) indicated that the paramagnetic signal recorded might be attributed to superoxide radicals. FR were absent in the AO/R1 group. Concurrently, the SOD activity revealed a significant tendency to return to the control level. FR appeared again in the AO/R2 group, mostly in the upper and middle lumbar regions, along with a decrease in SOD activity. No sample from the C group revealed FR. A significant increase in CD levels was observed in the thoracolumbar region only in the AO/R2 group. The temporary absence of FR in the AO/R1 group suggests activation of defense antioxidant mechanisms (e.g., specific enzymatic systems such as SOD), which might have been exhausted later.4. Changes in SOD activity similar to those observed in the thoracolumbar region, though less noticeable, occurred in the obviously noncompromised tissue (upper cervical region). This points to a kind of generalized reponse of the animal to aortic occlusion.5. Direct ESR spectroscopy revealed the presence of FR as well as their time course in the spinal cord during the early phase of ischemia/reperfusion injury and the inverse relationship between FR and SOD activity.     

The Clinical and Laboratory Studies of Superoxide Dismutase Activity in the Human Whole Blood with Early Gastric Cancer

In this study, we tried to evaluate the usefulness of Superoxide Dismutase (SOD) activity in detecting gastric cancer. SOD was determined according to M. Minami and H. Yoshikawa. a simple and inexpensive assay method. For II fibrognstroscopy and pathological proved gastric cancer cases, the average levels of SOD activity was found significantly lower than 30 patients with gastric diseases, 8 patients with atypical hyperplasia and 32 controls.     

Purified Human Neuromelanin, Synthetic Dopamine Melanin as a Potential Model Pigment, and the Normal Human Substantia Nigra: Characterization by Electron Paramagnetic Resonance Spectroscopy

Abstract: Neuromelanin is a poorly understood pigment that accumulates in catecholaminergic neurons during normal aging. Electron paramagnetic resonance spectroscopy, an especially effective technique for investigating melanins, is used in the present study to show unambiguously that neuromelanin is a melanin; however, it is not well modeled by synthetic dopamine melanin and thus is an atypical melanin. Some of the unusual features of neuromelanin can be explained by postulating two distinct sources for its free radicals, the dominant one possibly derived from a precursor containing sulfur. Examination of human substantia nigra by electron paramagnetic resonance spectroscopy during the purification of neuromelanin also demonstrates, contrary to some other studies, that a portion of the paramagnetic metal ions in this tissue are bound to the pigment in situ. Combined with previous histochemical data, these observations have implications for the mechanism through which neuromelanin accumulates in vivo and are consistent with its having a cytoprotective function under normal conditions, but a cytotoxic role at advanced ages and in patients with Parkinson's disease. Other results of this study show that homogenizing tissues during the purification of any natural pigment may cause contamination of the pigment by extraneous metal ions and that subsequent incubation in hot acid, though most effective in removing metal ions and hydrolyzing proteins, leads to degradation of melanin. A purification procedure using incubation in acid at room temperature, however, is well suited for identifying and characterizing unknown natural pigments by electron paramagnetic resonance spectroscopy.     

Studies on the Effects of Antioxidants and Inhibitors of Radical Generation on Free Radical Production in the Reperfused Rat Heart Using Electron Spin Resonance Spectroscopy

Reperfusion of the heart after a period of ischaemia can precipitate ventricular arrhythmias and lead to an exacerbation of tissue injury. Direct evidence to suggest the involvement of free radicals has been obtained using electron spin resonance (esr) spectroscopy and the spin trap N-tert. butyl-α-phenyl nitrone (PBN). In the present study, we have used esr spectroscopy and PBN to examine the individual effects of superoxide dismutase (SOD), catalase. allopurinol or desferal on radical production in the isolated. reperfused rat heart. A burst of radical production was observed in the control group during the first 5 minutes of reperfusion; the peak occurred during the first minute, when signal intensity had increased by almost 300%. but returned to the baseline by 15 minutes of reperfusion. The esr signals were consistent with the trapping of either alkoxyl or carbon-centered radicals (a_N = 13.6 and a_H = 1.56G). In the desferal-treated group, a burst of radical production was observed during the first five minutes of reperfusion; this was maximal during the second minute, when signal intensity had increased by almost 200%, but had returned to the baseline value by 30 minutes of reperfusion. In the SOD-treated group, a burst of radical production was observed during the first 10 minutes of reperfusion; signal intensity was maximal during the tenth minute of reperfusion, when signal intensity had increased by almost 200%. but had returned to the baseline value by 30 minutes of reperfusion. In the allopurinol- and catalase-treated groups, no significant burst of radical production could be detected. These data further support the concept that cytotoxic, oxygen-derived species are formed upon reperfusion and that hydrogen peroxide and/or hy-droxyl radicals, are likely to be involved.     

31P Nuclear Magnetic Resonance In Vivo Spectroscopy of the Metabolic Changes Induced in the Awake Rat Brain During KCN Intoxication and Its Reversal by Hydroxocobalamine

Radiofrequency surface coils were chronically implanted in rats, which were subsequently subjected to 31P nuclear magnetic resonance (NMR) investigations at 4.7 T. The implanted coil allowed study of the animals without need for anesthesia, which is a prerequisite for studies of normal brain metabolism. The animals may be kept in the NMR probe for several hours. During subsequent experiments, they may be placed in the same position, therefore allowing follow-up studies for periods as long as 2 months. This method has been used in the study of sublethal KCN intoxication. KCN, a cytochrome c oxidase inhibitor, induces a blockade of cell respiratory processes, which is reflected, in a dose-dependent manner, by a decrease in phosphocreatine content and pH and an increase in inorganic phosphate content, whereas ATP levels remain constant until high doses of KCN (6 mg/kg i.p.) are reached. 31P NMR allows the time course of these metabolic changes to be followed. For high KCN doses, a new peak, termed X, is observed, which is interpreted as being due to a pool of inorganic phosphate at very low pH (5.65), corresponding to a subset of cells that did not survive KCN injury. Hydroxocobalamine, a specific antidote of KCN, suppresses the metabolic changes due to 6 mg/kg of KCN.