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We examined the hypothesis that G-6-PD deficiency associated with fava bean ingestion confers resistance to malaria by studying the in vitro interactions between malaria parasites (Plasmodium falciparum),human erythrocytes with varying degrees of G-6-PD deficiency, and isouramil (IU), a fava bean extract that is known to cause oxidant stress and hemolysis of G-6-PD-deficient erythrocytes. Untreated G-6-PD-deficient and normal erythrocytes supported the in vitro growth of P. falciparumequally well. However, after pretreatment with IU, G-6-PD-deficient erythrocytes did not support parasite growth in vitro, whereas growth remained high in normal erythrocytes. Parasite growth was proportional to the G-6-PD activity of the IU-treated erythrocytes. In contrast, when parasitized erythrocytes were exposed to IU, parasites even in normal erythrocytes were destroyed. Ring forms were much less sensitive than late trophozoites and schizonts. The results suggest that there are two modes by which IU affects the development of P. falciparumand demonstrate in vitro that G-6-PD deficiency confers resistance against malaria under conditions of fava-bean-associated oxidant stress.

There is evidence that oxidative damage plays a causative role in Alzheimer′s disease and amyloid β protein (Aβ) toxicity. Iron is frequently a potent facilitator of free radical production due to its ability to mediate the conversion of H2O2to hydroxyl radicals via the Fenton reaction Or by virtue of hypervalent iron compounds. It is shown here that iron facilitates Aβ toxicity to cultured cells.

We examined the hypothesis that G-6-PD deficiency associated with fava bean ingestion confers resistance to malaria by studying the in vitro interactions between malaria parasites (Plasmodium falciparum), human erythrocytes with varying degrees of G-6-PD deficiency, and isouramil (IU), a fava bean extract that is known to cause oxidant stress and hemolysis of G-6-PD-deficient erythrocytes. Untreated G-6-PD-deficient and normal erythrocytes supported the in vitro growth of P. falciparum equally well. However, after pretreatment with IU, G-6-PD-deficient erythrocytes did not support parasite growth in vitro, whereas growth remained high in normal erythrocytes. Parasite growth was proportional to the G-6-PD activity of the IU-treated erythrocytes. In contrast, when parasitized erythrocytes were exposed to IU, parasites even in normal erythrocytes were destroyed. Ring forms were much less sensitive than late trophozoites and schizonts. The results suggest that there are two modes by which IU affects the development of P. falciparum and demonstrate in vitro that G-6-PD deficiency confers resistance against malaria under conditions of fava-bean-associated oxidant stress.

BACKGROUND: The healing-promotion property of saliva has been observed in the past, but its underlying mechanism has never been elucidated. We hypothesized a mechanism based on salivary proteins binding to redox active metal ions, rendering them nonactive in their capacity for free radical production. METHODS: Examination of this mechanism was conducted by comparing the redox activity of protein-rich saliva with protein-poor saliva. We also examined the redox activity mediated by these 2 kinds of saliva following the in vitro addition of iron, copper, and manganese. Saliva samples were analyzed for their redox activity by measuring the ascorbate-driven and saliva (diluted 1:2)-mediated conversion of salicylate to its 2,3- and 2,5-dihydroxybenzoates and catechol metabolites. RESULTS: The concentrations of salicylate metabolites formed by protein-rich saliva were significantly lower by 45% (P<.05), 66% (P<.01), and 54% (P<.05), respectively, when compared with those formed by proteinpoor saliva. The capacity of saliva in suppressing redox activity was found to be inversely related to the concentrations of iron and copper added (but not manganese), but correlated well with the protein content. When the highest concentrations of iron (15 μmol/L) and copper (10 μmol/L) were added to protein-rich saliva, the concentrations of salicylate metabolites produced were only 0.3% to 1% of those of non—saliva-containing controls (P<.01). However, when these concentrations of iron and copper were added to protein-poor saliva, significantly higher values of redox activity were detected, and the concentrations of the salicylate derivatives produced were 2.1% to 8.1% of those of non—saliva-containing controls (P<.01). In contrast, when the lowest concentrations of iron (2 μmol/L) and copper (0.1 μmol/L) were added, 2.8 to 4 times lower concentrations of salicylate derivatives were produced (P<.01). CONCLUSION: These results substantiate our hypothesis that saliva has a profound capacity for reducing redox activity rendered by transition metal ions, correlating well with its protein content.Arch Otolaryngol Head Neck Surg. 1997;123:989-993

Thioctic acid (TA) level and its antioxidant capacity were monitored by cyclic voltammetry (CV), in solution and in human plasma. A linear correlation between the anodic wave current Ia at 900 mV and [TA] was obtained. This indicates that TA, commonly found in human plasma, is an antioxidant component characterized by a potential E1/2=900 mV and constitute, at least in part, the second anodic wave of the CV tracing. When plasma from diabetic patients was analyzed by CV, those patients under treatment with TA showed higher Ia than others not taking the drug. When the patients under treatment with TA were divided into groups according to the severity of their nephropathy, or according to the severity of their total complication count, Ia levels representing TA were significantly higher in those patients with more severe complications.

The mechanism of enzymatic inactivation of purified and membrane-bound acetylcholine esterase by ascorbate and copper was investigated. While the exposure of the enzyme to either ascorbate or copper did not cause enzymatic inactivation, the incubation of the enzyme with a combination of both ascorbate and copper resulted in a loss in acetylcholine esterase activity, which was time dependent. The enzymatic inactivation required either molecular oxygen or hydrogen peroxide under anaerobic conditions. Scavengers of hydroxyl radicals at concentrations of up to 100 mM did not provide protection to acetylcholine esterase. Only mannitol at very high concentrations (above 1 M) efficiently prevented the inactivation of the enzyme. The kinetics of the aerobic oxidation of reduced ascorbate in the presence of acetylcholine esterase and copper closely followed the rate of enzyme inactivation. Addition of the chelating agents EDTA and diethylenetriaminepentaacetic acid prevented both the oxidation of ascorbate and the inactivation of the enzyme. In the presence of low concentrations of histidine (0.5-2.0 mM), which forms high affinity complexes with copper, the rate of ascorbate oxidation was similar to that recorded in its absence. On the other hand, no enzyme inactivation was indicated in the presence of histidine. Low temperature EPR measurements have demonstrated the binding of copper to the enzyme, and have shown the reduction of the cupric enzyme to the corresponding cuprous complex. In view of these results, a general “site-specific” mechanism for biological damage can be offered, in which copper(II) ions are bound to enzymes or other biological macromolecules. Ascorbate plays a dual role: it reduces the cupric complex to the corresponding cuprous state and serves as a source for H2O2, which, in turn, reacts with the reduced copper complex, in a Fenton reaction. In this reaction, secondary hydroxyl radicals are site specifically formed, and react preferentially with the protein, at the site of their formation, causing its inactivation. This mechanism is analogous to that previously proposed (Samuni, A., Chevion, M., and Czapski, G. (1981) J. Biol. Chem. 256, 12632-12635) for the enhancement of the biological damage caused by superoxide in the presence of copper.

Hemoglobin A and hemoglobin Kansas can exist in alternative forms in which the affinity of heme iron for NO is mediated either by pH or the presence of allosteric modifiers. In this study, we show that the superhyperfine (SHF) pattern observed in the EPR which arises from the heme iron‐NO complex can be correlated with a change in the affinity of the ligated tetrameric protein for NO. When a combination three‐line and nine‐line SHF pattern is observed, the nitrosyl hemoprotein is in a low affinity form while a nine‐line pattern alone is an expression of high affinity. A theory is presented relating SHF patterns to a molecular orbital picture of the nitrosyl heme complex. It is suggested that the change of SHF pattern correlated with an alteration of the affinity state of ligated hemoglobin is related to a structural change of the proximal imidazole ligand to the heme.

Exposure of albumin to Cu(II) (10-100 microM) and ascorbate (0.1-2 mM) results in extensive molecular modifications, indicated by decreased fluorescence and chain breaks. The rate of utilization of molecular oxygen and ascorbate as a function of Cu(II) concentration is non-linear at copper/albumin ratios of greater than 1. It appears that Cu(II) bound to the tightest albumin-binding site is less available to the ascorbate than the more loosely bound cation. SDS/polyacrylamide-gel electrophoresis reveals new protein bands corresponding to 50, 47, 22, 18 and 3 kDa. For such a cleavage pattern, relatively few (approximately 3) and rather specific chain breaks occurred. Repeated addition of portions of ascorbate to the albumin/Cu(II) mixture results in increased intensity of the new bands. The absence of Cu(II) or the presence of metal chelating agents is inhibitory. There was no evidence of intermolecular cross-linking or of the formation of insoluble, albumin-derived, material. A mechanism is proposed wherein the loosely bound Cu(II) participates in a Fenton-type reaction. This generates OH. radicals, which rapidly inter-react with the protein and modify it in a ‘site-specific’ manner.

Direct evidence for substantial mobilization of copper in the coronary flow immediately following prolonged, but not short, cardiac ischemia is presented. In the first coronary flow fraction (CFF) of reperfusion (0.15 ml), after 35 min of ischemia, the level of copper (as well as of iron) was 8- to 9-fold higher than the preischemic value. The levels in subsequent CFFs decreased and reached the preischemic value, indicating that both metals appear in a burst at the resumption of coronary flow. When the first CFF was used in a reaction mixture containing ascorbate and salicylate, the latter underwent chemical hydroxylation and was converted to its dihydroxybenzoate derivatives. Likewise, this CFF promoted the ascorbate-driven DNA degradation. Subsequent 150 CFFs were serially collected and demonstrated low activities. Following 18 min of ischemia, the copper level in the first CFF of reperfusion was only 15% over the preischemic value. In contrast, the mobilization of iron into coronary flow was significant but markedly lower than after 35 min. The levels of copper and the redox activity of the first CFF correlated well with the degree of loss of cardiac function, after 18 and 35 min of ischemia, respectively. After 18 min of ischemia, cardiac function was about 50% and the damage is considered reversible, whereas after 35 min the functional loss exceeded 80% and is considered irreversible. These results are in accord with the causative role that copper and iron can play in heart injury following ischemia, by virtue of their capacity to catalyze the production of hydroxyl radicals, and could lead to the development of new modalities for intervention in tissue injury.

The zinc-desferrioxamine (Zn-DFO) complex is considered to be more permeative into parasitized erythrocytes than is the metal-free DFO. The former may penetrate the cell and exchange its bound zinc for ferric ions, rendering the iron unavailable for vital parasite functions. The effects of these compounds on the in vitro development of Plasmodium falciparum are compared. The results indicate that Zn-DFO is superior to DFO, especially at concentrations below 20 microM, as shown by decreased levels of hypoxanthine incorporation, lower levels of parasitemia, and interference with the life cycle of the parasite. At low concentrations, DFO even enhanced parasite growth. Such an enhancement was not observed following exposure to Zn-DFO. Experiments in which the compounds were removed from the cultures indicated that parasites treated with Zn-DFO are less likely to recover at a later stage. Since DFO has already been used in humans for the treatment of malaria, its complex with zinc, which is more effective in vitro, should also be examined in vivo.

The role of iron ions in paraquat toxicity was studied in bacterial system. We show that addition of ferrous iron led to an enhancement of the bacterial killing, whereas addition of chelating agents, such as nitrilotriacetate and desferrioxamine, markedly reduced, up to a total abolishment, the toxic effects. The calculated rates of bacterial killing are proportional to both paraquat and iron concentrations, and conform to the rate equation: dN/dt = -k[paraquat] [Fe2+]. The killing constant for iron, k, is 24-fold smaller than the corresponding value for copper. Mannitol, an OH. scavenger, has a partial protective effect: 15-35% at concentrations range of 1-50 mM, respectively. Histidine, on the other hand, provided a more efficient protection that may be due to a combination of various effects. Induction of endogenous superoxide dismutase and catalase provided partial protection (about 25%). These findings, together with an earlier study on the role of copper in paraquat toxicity (Kohen, R., and Chevion, M. (1985) Free Rad. Res. Commun. 1, 79-88) indicate that transition metals play a central catalytic role in the production of the deleterious effects of paraquat, probably by redox cycling and producing OH. via the site-specific Fenton reaction.

The role of reactive oxygen species (ROS) generated by polymorphonuclear leucocytes (PMNs) in the host response against malaria was investigated. Non-activated human PMNs were added to cultures of P. falciparum in microtitre cells. Parasite viability was evaluated by the incorporation of radioactive hypoxanthine. Using PMN/RBC = 1/150 (starting parasitemia was 1+) the incorporation on the second day in culture was only 61+ of the control cultures. An effect could be observed already after two hours of incubation (30+ reduction at a 1/50 PMN/RBC ratio). A direct contact between the effector and target cells was obligatory for the expression of the damage.Parasites within G6PD-deficient erythrocytes were more sensitive to the PMNs than normal parasitized erythrocytes. This difference could be attributed to the production of reactive oxygen intermediates in the experimental system, since G6PD-deficient erythrocytes are generally more sensitive to oxidant stress.Salicylic acid was used as a scavenger and reporter molecule for hydroxyl radical fluxes. It is converted to the corresponding dihydroxybenzoic acid derivatives, which could be detected by HPLC. Uninfected NRBC or parasitized erythrocytes containing young ring forms could trigger the PMNs to produce much less ROS than the mature forms of the parasites. Other factors associated with PMNs may inactivate the parasites, such as phagocytosis, lysosomal enzymes or degradation toxic products of the PMNs. However our results indicate that increased oxidative stress induced by PMNs interfere with the growth of P. falciparum and could play a role in human evolution of abnormal erythrocytes.

The essential mediatory role of copper and iron in paraquat-induced biological damage has been recently demonstrated. It was postulated that these transition metals undergo cyclic redox reactions and serve as centers for repeated production of hydroxyl radical, which are the ultimate deleterious agents. Additionally, we had presented evidence indicating efficient protection against paraquat toxicity by agents commonly employed (chelators, chemical scavengers, and protecting enzymes).

The pulse radiolysis of solutions of adult human methemogolbin was used in order to reduce a single heme iron within the protein tetramers. The valence hybrids thus formed were reacted with oxygen. Kinetics of the reactions were studied. The effects of pH and inositol hexaphosphate were examined. The kinetics of the ligation of oxygen to stripped valence hybrids showed a single phase behavior at the pH range 6.5 to 9. As the pH was lowered below 6.5, a second, slower phase became apparent. In the presence of inositol hexaphosphate, above pH 8, the kinetics of oxygen binding was of a single phase. As the pH was lowered, a transition to a second, slower phase was noticed. Below pH 7, the slower phase was the only detectable one. The analysis of the relative contribution of the faster phase to the total reaction as a function of the pH showed a typical transition curve characterized by a pK = 7.5 and a Hill parameter n = 2.9. On this basis, it is concluded that human adult stripped methemoglobin resides in an R quarternary structure, while the presence of IHP stabilizes the T structure at pH below 7.5. This transition between the quaternary structures of methemoglobin cannot be accounted for by the switch between the high spin and the low spin states of the ferric iron. This switch of spin state takes place at pH greater than 8.2.

Background. Reactive oxygen species are an important mediator in isoflurane-induced myocardial preconditioning. However, hydroxyl radicals are also released during reperfusion after regional ischaemia. The purpose of the present study was to test whether ischaemic preconditioning and isoflurane would influence the production of hydroxyl radicals during reperfusion. Methods. After i.v. administration of salicylate 100 mg kg−1 and a 30 min stabilization period, New Zealand White rabbits were subjected to 40 min of regional myocardial ischaemia and 2 h of reperfusion. Ischaemic preconditioning was elicited by 5 min ischaemia followed by 10 min reperfusion (before the 40 min ischaemia). In another group, isoflurane (2.1%) was administered for 30 min, followed by 15 min washout, before the long ischaemia. Area at risk and infarct size were assessed by blue dye injection and tetrazolium chloride staining. We quantified the level of OH-mediated conversion of salicylate to its dihydrobenzoate derivatives (2,3- and 2,5-DHBAs). Normalized values of the DHBAs (ng DHBA per mg salicylate) were calculated. Results. Mean (se) infarct size was 57 (6)% of the risk area in the untreated controls. This was significantly smaller in the ischaemic preconditioning and isoflurane groups: 22 (5) and 23 (6)% respectively. At 10 min of reperfusion, ischaemic preconditioning limited the mean increase in 2,3-DHBA to 24% from baseline, compared with 81% in control and 74% in the isoflurane group. Normalized 2,5-DHBA was maximally increased by 75% in the untreated group, 4 min after reperfusion. Ischaemic preconditioning significantly inhibited this increase (24% increase from baseline, P<0.01). However, the increase observed in the isoflurane group was not different from control (71%). Conclusions. As already known, ischaemic preconditioning and isoflurane markedly reduced infarct size. However, only ischaemic preconditioning decreased postischaemic production of hydroxyl radicals. These different effects suggest different protective mechanisms at the cellular level.

The addition of isouramil,an aglycon derived from broad peans,has the capacity to form hydrogen peroxide and free radical metabolites.Hydrogen peroxide, if not detoxified by the glutathion peroxidase system,can cause damage to RBC membranes by lipid peroxidation.MDA,a breakdown product of lipid hydroneroxides in biological systems,causes similar peroxidation of endogenous membrane phospholipids as well as a decrease in band 1 and 2 of spectrin and an increase in high molecular weight protein polymers.This study was Hesigned to investigate the effect of these alterations on RBC microrheologic behavior in vitro.Human PBC were exposed to ISO(1mM)and MDA(5,10,20µM).Using a cone-plate Rheoscope RBC deformation was observed and measured over a wide shear Btress range(2.5-500 dyn/cm2).Without changes in cell shape pr volume cell deformation decreased significantly at low shear stresses for all experiments and over the whole shear stress range for cells treated with 10 or 20 µM KDA.The addition of glucose(5mM)during the incubation period prevented the ISO induced changes in deformation.The results suggest that the polymerisation of membrane components may enhance sequestration of aging cells or cells exposed to autooxidizable substances.Supported by DFG grant PF 184/1-1