The role of free radical reactions with haemoglobin and thalassaemia

It is well known that all living systems depend on iron to transport (haemoglobin), store (myoglobin) and utilize (cytochromes, cytochrome oxide) oxygen for respiration. Iron is an essential component in the active sites of the enzyme that protects against oxidation, such as the iron superoxide dismutase, in bacteria and plants. In normal human plasma almost all iron loading of transferrin is 20-30% maximum. In this presentation we review and summarize recent developments in our understanding of iron transport and storage in living systems.     

Effect of galactose on free radical reactions of polymorphonuclear leukocytes.

To account for impaired bactericidal activity of polymorphonuclear leukocytes during galactosemia, we have investigated the effects of galactose upon free radical reactions associated with the oxygen-dependent killing mechanism of guinea pig polymorphonuclear leukocytes. Millimolar levels of galactose, which are encountered in the circulation of infants with galactosemia, did not affect the chemical reaction of Superoxide anion with either ferricytochrome c or nitroblue tetrazolium. Galactose did, however, significantly inhibit the reaction of hydroxyl radical with methional, and we propose that such hydroxyl radical removal would be deleterious to normal bactericidal activity. Additional experiments demonstrated that polymorphonuclear leukocytes incubated in the presence of galactose reduced less ferricytochrome c than polymorphonuclear leukocytes incubated without galactose. These results imply that galactose impairs the cellular release of Superoxide anion, which would further disable bactericidal activity.     

The role of carbon dioxide in free radical reactions of the organism

Carbon dioxide interacts both with reactive nitrogen species and reactive oxygen species. In the presence of superoxide, NO reacts to form peroxynitrite that reacts with CO2 to give nitrosoperoxycarbonate. This compound rearranges to nitrocarbonate which is prone to further reactions. In an aqueous environment, the most probable reaction is hydrolysis producing carbonate and nitrate. Thus the net effect of CO2 is scavenging of peroxynitrite and prevention of nitration and oxidative damage. However, in a nonpolar environment of membranes, nitrocarbonate undergoes other reactions leading to nitration of proteins and oxidative damage. When NO reacts with oxygen in the absence of superoxide, a nitrating species N2O3 is formed. CO2 interacts with N2O3 to produce a nitrosyl compound that, under physiological pH, is hydrolyzed to nitrous and carbonic acid. In this way, CO2 also prevents nitration reactions. CO2 protects superoxide dismutase against oxidative damage induced by hydrogen peroxide. However, in this reaction carbonate radicals are formed which can propagate the oxidative damage. It was found that hypercapnia in vivo protects against the damaging effects of ischemia or hypoxia. Several mechanisms have been suggested to explain the protective role of CO2 in vivo. The most significant appears to be stabilization of the iron-transferrin complex which prevents the involvement of iron ions in the initiation of free radical reactions.     

Some bold evolutionary predictions for the future of mating in humans

Why are many human populations presently ‘imploding’ with below‐replacement fertility? Why are more and more young adults in these societies choosing to remain single and/or childless? Based on first principles of evolutionary theory, predictions can be derived for changes over time in the relative frequency distributions of four traits in humans proposed as the most direct determinants of the propensity to mate and reproduce: attractions to sex, legacy, leisure and parenting. In the past, high fitness was most profoundly determined by strong sex drive and strong legacy drive, especially in males. Female fertility was largely controlled by dominant males, who were then free to engage in attractions to both leisure and legacy through ‘memes’ (as well as through genes, or offspring) without any penalty on fitness. Natural selection in the past, therefore, neither strongly favoured nor strongly disfavoured any particular intrinsic female inclinations or preferences that might affect offspring production. The recent, widespread, and continuing rise in the empowerment of women, however, defines a dramatically different contemporary selection regime, where women are now free to indulge in their evolved attractions to leisure and legacy through memes inherited from predecessors, both of which represent compelling distractions from parenthood. The implications for the future survival of marriage and parenthood as cultural institutions look dismal in the short term, but promising in the long term.     

Competitive kinetics in free radical reactions of cinnamic acid derivatives. Influence of phenoxyl radicals reactions

Relative rates of consumption of caffeic, ferulic and sinapic acids by 2,2'-azobis(2-amidine propane) derived peroxyl radicals has been measured in parallel experiments employing a single substrate and in competitive experiments. Rates of consumption measured in independent experiments at low substrate concentrations (first order limit) follow the order: sinapic > ferulic > caffeic. In agreement with this, in competitive experiments employing simultaneously sinapic and caffeic acid the former compound is consumed considerably faster. On the other hand, in competitive experiments employing ferulic and caffeic acids over a wide range of experimental conditions, caffeic acid is consumed considerably faster than ferulic acid, a result that contrasts with that obtained when both compounds are reacted independently. These apparently anomalous results are interpreted in terms of secondary reactions of the phenol-derived radicals. In particular, hydrogen transfer among phenoxyl radicals and the phenols and fast reactions (disproportionation) of caffeic acid derived radicals could explain these discrepancies.     

Competitive kinetics in free radical reactions of cinnamic acid derivatives. Influence of phenoxyl radicals reactions

Relative rates of consumption of caffeic, ferulic and sinapic acids by 2,2′-azobis(2-amidine propane) derived peroxyl radicals has been measured in parallel experiments employing a single substrate and in competitive experiments. Rates of consumption measured in independent experiments at low substrate concentrations (first order limit) follow the order: sinapic > ferulic > caffeic. In agreement with this, in competitive experiments employing simultaneously sinapic and caffeic acid the former compound is consumed considerably faster. On the other hand, in competitive experiments employing ferulic and caffeic acids over a wide range of experimental conditions, caffeic acid is consumed considerably faster than ferulic acid, a result that contrasts with that obtained when both compounds are reacted independently. These apparently anomalous results are interpreted in terms of secondary reactions of the phenol-derived radicals. In particular, hydrogen transfer among phenoxyl radicals and the phenols and fast reactions (disproportionation) of caffeic acid derived radicals could explain these discrepancies.     

The hydroxyl free radical reactions of ascorbyl palmitate as measured in various in vitro models.

The OH(*) free radical scavenging properties of ascorbyl palmitate (AP), water-solubilized in the presence of a surfactant (Brij 35), were tested in various systems: (1) The inhibition of polymerization of bovine serum albumin by OH(*) free radicals generated by the Fenton reaction indicated AP exerts a considerable protective effect against polymerization by scavenging the OH(*) free radicals. (2) ESR spin trapping comparisons of DMPO with AP were conducted. Using the Fenton reaction as a source of OH(*) free radicals, AP was 1 order of magnitude faster in scavenging these radicals than DMPO. (3) Oxidative modification of BSA by (60)Co-gamma irradiation of 80 krad, results in a strong increase in protein carbonyl content. AP inhibits carbonyl formation very efficiently, indicating that AP may be utilized as a biological OH(*) free radical scavenger in human therapy.     

Depolymerization of xanthan by iron-catalysed free radical reactions

It is demonstrated using gamma-radiolytic and photolytic techniques that, when O₂?- is produced in the presence of catalytic amounts of iron(II) and iron(III) complexes depolymerization of xanthan in aqueous solution occurs. The use of these techniques also allows a quantitative measurement of the efficiency of conversion of O₂^?- to ?OH in these systems to be made, with the assumption that ?OH is the most likely depolymerizing species present. Using pulse radiolysis and oxidative-reductive depolymerization, the effects of some common anions on the degradation process have been correlated with the reactivities of their related free radicals with xanthan. The effects of other parameters such as ionic strength and temperature on xanthan depolymerization are also detailed     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.     

Kinetic chemiluminescence as a method for study of free radical reactions

The review gives concepts of analysis of kinetics of complex reactions with the participation of free radicals. The concepts are based on the comparison of kinetic curves of chemiluminescent reactions in the presence of a physical enhancer coumarin C-525 with the simulated kinetic functions. This method was applied to the investigation of the mechanism of a branched-chain reaction of lipid peroxidation in biological and phospholipid membranes as well as to the effect of antioxidants and determination of their activities. It was also used for studying the reaction of formation of free radicals in the complex of cytochrome c with cardiolipin. This reaction plays a key role in the initiation of apoptosis.     

Consequences of chemical modifications on the free radical reactions of human hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are candidates for use as blood substitutes and resuscitation fluids. We determined that HBOCs of specific types differ in their ability to generate or interact with free radicals. The differences do not correlate with oxygen affinity. Detailed comparisons with unmodified human hemoglobin, HbA0, were carried out with two cross-linked derivatives: HbA-FMDA, produced by the reaction of human oxyhemoglobin with fumaryl monodibromoaspirin, and HbA-DBBF, produced by the reaction of human deoxyhemoglobin with bis(3,5-dibromosalicyl) fumarate. Both derivatives had lower oxygen affinity than unmodified HbA0. As previously reported, exposure of oxyhemoglobin to H2O2 causes generation of free radicals capable of generating formaldehyde from dimethyl sulfoxide. Relative to the reaction catalyzed by 50 microM HbA (18.0 +/- 3.5 nmol/30 min/ml), the formaldehyde formation was roughly 70% for HbA-DBBF and 50% for HbA-FMDA under comparable conditions. More profound differences are exhibited at lower hemoglobin concentrations. Spectral changes of the HBOCs during the reaction differ qualitatively and occur at different rates. The HBOCs also differ in rates of hemoglobin-catalyzed NADPH oxidation and aniline hydroxylation, reactions mediated by reactive oxygen species. These results show that stereochemical differences brought about by chemical cross-linking alter the ability of HBOCs to generate radicals and to react with activated oxygen species. These studies also show that the ability of hemoglobin to produce activated species of oxygen can be enhanced or suppressed independently of oxygen affinity.     

Kinetic chemiluminescence as a method for the study of free radical reactions

The review gives concepts of analysis of kinetics of complex reactions with the participation of free-radicals. The concepts are based on the comparison of the kinetic curves of the chemiluminescent reactions in the presence of a physical enhancer coumarin C-525 with the simulated kinetic functions. This method was applied to the investigation of the mechanism of a branched-chain reaction of lipid peroxidation in biological and phospholipid membranes as well as to the effect of antioxidants and determination of their activities. It was also used for studying the reaction of the formation of free radicals in the complex of cytochrome c with cardiolipin. This reaction plays a key role in the initiation of apoptosis.     

A pulse radiolysis study of some free radical reactions with erythrocyte membranes.

The reactions of the free radicals eaq- minus, OH and Br2- minus with haemoglobin-free erythrocyte ghost membranes have been studied by producing the radicals by pulse radiolysis and monitoring their reactions by optical spectroscopy. Hydrated electrons react rapidly with the membrane, but no attack at disulphide links was observed. Hydroxyl radical attack produced transient species absorbing weakly in the ultraviolet, which may arise from carbohydrate residues, such as N-acetyl neuraminic acid and N-acetyl glucosamine, on the membrane surface. No evidence was obtained for OH attack at ring-containing amino acid residues of the protein component. The Br2- minus radical, a more selective electrophile than OH, reacted only slowly with erythrocyte ghosts. Solubilization of the membranes with dodecylsulphate or digestion with alkali exposed protein containing tyrosine and tryptophan residues which reacted with Br2- minus. These results support other evidence for the absence of reactive protein at the membrane surface.     

The interaction of Rubia cordifolia with iron redox status: a mechanistic aspect in free radical reactions.

The present publication investigates the antioxidant property and mechanistic aspect of alcoholic extract of R. cordifolia. The extract of R. cordifolia has shown significant inhibitory effect on FeSO4 induced lipid peroxidation. Study with iron redox status showed that R. cordifolia extract reduced or oxidixed; Fe3+ or Fe2+ respectively, in a dose dependent manner. Results with superoxide anion (O2-.) and hydroxyl radical (OH.), showed no radical scavenging activity. The alcoholic extract significantly maintains the reduced glutathione content both in time and dose dependent manner. It also reduced the rate of depletion of reduced glutathione (GSH) level in presence of ferrous sulphate (FeSO4) and cumene hydroperoxide (CHP). On the basis of these observations, it can be concluded that the antioxidant property of R. cordifolia is due to a direct interaction with iron.