Studies on vitamin E. 5. Lipid peroxidation in dialuric acid-induced haemolysis of vitamin E-deficient erythrocytes.
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Barbiturates↗
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Oxidation of 5-hydroxypyrimidine nucleosides to 5-hydroxyhydantoin and its alpha-hydroxy-ketone isomer.
The reaction of hydroxyl radicals with 2'-deoxycytidine (dCyd), as well as the decomposition of dCyd radical cations, leads to a complex mixture of oxidation products in aqueous aerated solutions. The oxidation of dCyd gives products with a relatively low oxidation potential that are highly susceptible to further oxidation, including 5-hydroxy-2'-deoxycytidine (5-oh-dCyd) and 5-hydroxy-2'-deoxyuridine (5-oh-dUrd). Previously, we showed that the oxidation of 2'-deoxyuridine (dUrd) involves the formation of dialuric acid and isodialuric acid intermediates, followed by ring contraction to N1-(2-deoxy-beta-D-erythro-pentofuranosyl)-5-hydroxyhydantoin (5-oh-dHyd). In this work, we have examined the oxidation of 5-oh-dCyd and 5-oh-dUrd in greater detail. The oxidation of these substrates by Br2 led to a similar profile of intermediate and stable products indicating that the dialuric and isodialuric acid derivatives of dCyd largely undergo deamination before they transform into 5-oh-dHyd. Analysis of the final mixture of oxidation products by HPLC revealed the formation of two novel products. On the basis of NMR and MS, these products were identified as the diastereomers of N1-(2-deoxy-beta-D-erythro-pentofuranosyl)-5-hydroxyimidazolidine-2,5-dione (iso-4-oh-dHyd). These products arise from alpha-hydroxy-ketone isomerization of 5-oh-dHyd. The isomerization of 5-oh-dHyd to iso-4-oh-dHyd was reversible, and each diastereomer produced a specific diastereomer of the other structural isomer. The rate of isomerization was accelerated in going from pH 5 to pH 9, whereas all isomers decomposed at higher pH. In contrast, interconversion between each pair of diastereomers was minor. Thus, we conclude that the oxidation of 5-oh-dCyd or 5-oh-dUrd gives a mixture of four isomers of 5-oh-dHyd and iso-4-oh-dHyd as final products. The biological consequences of dCyd oxidation may ultimately depend on the effects of these products.
Chromatography, High Pressure Liquid↗
Transformation of barbituric acid into alloxan by hydroxyl radicals: interaction with melatonin and with other hydroxyl radical scavengers.
Barbituric acid (2,4,6-pyrimidinetrione) can be transformed by a non-enzymatic hydroxylation into alloxan (2,4,5,6-pyrimidinetetrone). This transformation can be used as a reaction indicating the formation of hydroxyl radicals (.OH). This conversion was detected using HPLC. Formation of .OH was demonstrated by electron spin resonance (ESR) spectroscopy combined with spin-trapping techniques. It was shown that .OH generated via the Fenton reaction abstracts first a hydrogen atom from barbituric acid (BA) and forms intermediately a paramagnetic derivative of BA. After a second attack by another .OH, the BA radical is transformed into dialuric acid (DA), which autoxidizes via the alloxan radical (.ALX) to ALX. Superoxide radicals (.O2-) are formed during autoxidation of DA and.ALX. They are able to regenerate ferrous ions. As a result, traces of iron salts are capable of catalyzing the conversion of large amounts of BA into ALX. Several scavengers of .OH were tested with regard to their efficiency in preventing the transformation of BA into ALX. Of all the scavengers analyzed, melatonin was shown to be one of the most potent compounds.
Alloxan↗
Dietary sesame lignans decrease lipid peroxidation in rats fed docosahexaenoic acid.
We have previously reported that dietary sesamin and sesaminol, major lignans of sesame seed, elevate the alpha-tocopherol concentration and decrease the thiobarbituric acid reactive substance (TBARS) concentration in the plasma and liver of rats. In this study, the effects of dietary sesamin and sesaminol on the lipid peroxidation in the plasma and tissues of rats fed docosahexaenoic acid (DHA, 22:6 n-3) were examined. Male Wistar rats (4-wk-old) were divided into the following six experimental groups: control group, fed a basal diet: sesamin group, fed a diet with sesamin (2 g/kg); sesaminol group, fed a diet with sesaminol (2 g/kg); DHA group, fed a diet containing DHA (5 g/kg); DHA + sesamin group, fed a diet containing DHA with sesamin; and DHA + sesaminol group, fed a diet containing DHA with sesaminol. Each diet contained either 0.01 or 0.05 g D-alpha-tocopherol/kg, and the rats were fed the respective experimental diet for 5 wk. The dietary DHA elevated the TBARS concentration and also increased the red blood-cell hemolysis induced by the dialuric acid. The dietary sesamin and sesaminol lowered the TBARS concentrations and decreased the red blood hemolysis. The dietary sesamin and sesaminol elevated the alpha-tocopherol concentrations in the plasma, liver, and brain of the rats fed a diet with or without DHA. These results suggest that dietary sesame lignans decrease lipid peroxidation as a result of elevating the alpha-tocopherol concentration in rats fed DHA.
Animals↗
The behavior of alloxan in the Hill reaction.
Hill activity induced by alloxan is characterized by non-stoichiometric oxygen evolution and cyclic electron flow due to autoxidation of dialuric acid and alloxantin, the reduction products of alloxan.
Alloxan↗
Alloxan acts as a prooxidant only under reducing conditions: influence of melatonin.
Depending on the availability of suitable reducing agents, alloxan can be either a prooxidant or an antioxidant. Alloxan and its reduced derivative, dialuric acid, act as a redox couple, driven by reduced glutathione (GSH) or L-cysteine, generating in vitro in the presence of oxygen, both superoxide radical and hydrogen peroxide. The production of superoxide radicals was shown by the appearance of lucigenin chemiluminescence (CL) as well as by the generation of formazan from nitroblue tetrazolium (NBT). The lucigenin CL as well as the NBT reduction was inhibited by superoxide dismutase and partially by catalase. Melatonin inhibited alloxan-mediated CL. In contrast, in the absence of reducing agents, alloxan is a scavenger of superoxide radicals formed by other reactions. Because of the high content of reducing compounds in the cell (e.g. glutathione), it is suggested that alloxan acts in vivo mainly as a generator of reactive oxygen species.
Alloxan↗
Thiol-group reactivity, hydrophilicity and stability of alloxan, its reduction products and its N-methyl derivatives and a comparison with ninhydrin.
The diabetogenic agent, alloxan, is a hydrophilic and chemically unstable compound. The logarithm of the octanol/water partition coefficient of alloxan was found to be -1.86; its half-life at pH 7.4 and 37 degrees in phosphate buffer was 1.5 min. The partition coefficients and half-lives of the alloxan reduction products, alloxantin and dialuric acid, were very similar to those of the parent compound; N-methylalloxan and N,N'-dimethylalloxan were less hydrophilic but more unstable. Ninhydrin was found also to be hydrophilic although this compound, in contrast to alloxan and its derivatives, was quite stable in aqueous solution. Alloxan and its N-methyl derivatives were reduced by thiols and in the presence of glutathione and cysteine, rapid redox cycling occurred, with formation of 'active oxygen' species; no such reaction was observed, however, with ninhydrin. Comparatively slow redox cycling was recorded with alloxan derivatives and dithiothreitol although rapid cycling occurred with ninhydrin and this dithiol. Such differences may explain why ninhydrin does not share with alloxan a selective toxic effect upon the pancreatic B-cell.
Alloxan↗
The biochemical modes of action of vitamin e and selenium: A hypothesis.
In this paper recent experimental work on rat liver is discussed which is considered to indicate that a primary function of vitamin E in vivo may be to inhibit the oxidation of selenide-containing proteins present in mitochondria and in smooth endoplasmic reticulum. On the basis of molecular model building studies, it is also suggested that the well-known nutritional relationships between vitamin E and dietary polyunsaturated lipids may be due to the occurrence within normal membranes of specific complexes between the vitamin and some of the molecules of polyunsaturated phospholipids. Without the vitamin, membranes may have an abnormally high permeability and they may be subject to degradation by endogenous phospholipases in vivo, as well as being abnormally susceptible to damage in vitro by dialuric acid and hydrogen peroxide.
Journal Article↗
Formation of compound 305 requires the simultaneous generation of both alloxan and GSH radicals.
This in vitro study investigates the conditions under which "compound 305" is formed. Using HPLC, ESR as well as UV spectroscopy, "compound 305" was largely separated and characterized. It has an absorption peak at 314 nm, which changes after reoxygenation to shorter wavelengths within hours. The retention time of "compound 305" amounts to 10.93 +/- 0.042 min. The formation of "compound 305" does not depend on alloxan (ALX) or reduced glutathione (GSH), but most likely on the steady-state concentration of the paramagnetic derivatives of both reactants (ALX* and GS*). The alloxan radical (ALX*) is formed by either a one-electron transfer from e. g. GSH to alloxan or oxidation of dialuric acid. The concentration of the ALX* was determined to be 12 +/- 3.6 micromol/l using the stable ultramarine radical as an ESR standard. ALX* is stable only under anaerobic conditions. It disappears within 2 min in air. Since formation of "compound 305" needs both ALX* as well as GS*, which are also necessary for the generation of reactive oxygen species (ROS), it is assumed that formation of "compound 305" diminishes the toxicity of alloxan.
Alloxan↗
Relative activity of alpha-tocopherol and gamma-tocopherol in preventing oxidative red cell hemolysis.
The purpose of this study was to compare the antioxidant activities of alpha-tocopherol and gamma-tocopherol in protecting the red cell membrane against lipid peroxidation. Tocopherols were incorporated into the red cell membrane by incubating cells with solutions of the tocopherols in bovine albumin. The cells were then washed and subjected to the dialuric acid hemolysis test. Analysis of variance of the response curves revealed that gamma-tocopherol had 38% of the activity of alpha-tocopherol. No evidence was found for an interaction between the two tocopherols when present in the red cell membranes simultaneously.
Animals↗
Relative antioxidant effectiveness of alpha-tocopherol and gamma-tocopherol in iron-loaded rats.
The relative antioxidant effectiveness of RRR-alpha-tocopherol and d-gamma-tocopherol against in vivo lipid peroxidation in vitamin E-depleted, iron-loaded rats was assessed by measurement of expired pentane. Rats fed a vitamin E-deficient diet were each administered 103 +/- 2 mg of iron as iron dextran over a 4-week period. After 3 weeks, their erythrocytes were 96.9 +/- 0.6% hemolyzed by dialuric acid. After 6 weeks, the rats exhaled 22.4 +/- 3.4 pmol pentane/(100 g body weight . minute). Groups of 4 rats each were then fed varying levels of RRR-alpha- and d-gamma-tocopherol for 2 weeks, after which the pentane levels were directly related to the dietary tocopherol content. Covariance analysis of the log of pentane production versus the log of dietary tocopherol showed the relative antioxidant effectiveness of 1:0.31 for alpha-tocopherol:gamma-tocopherol. In an independent estimation of relative antioxidant effectiveness, covariance analysis of the log of lipid soluble fluorophores in the spleens of the rats versus the log of dietary tocopherol showed a ratio of 1:0.37 for alpha-tocopherol:gamma-tocopherol. Regression analysis showed the fluorophores also to be correlated with the integrated amount of pentane produced over the 7-week experiment (r = 0.84, P less than 0.001). gamma-Tocopherol was more effective as an in vivo antioxidant than has been reported for its inhibition of vitamin E-deficiency syndromes.
Animals↗
Inhibition of biogenic amine uptake by hydrogen peroxide: a mechanism for toxic effects of 6-hydroxydopamine.
Hydrogen peroxide, dialuric acid, or 6-hydroxydopamine inhibited the uptake of dopamine, norepinephrine, and serotonin into rat brain synaptosomal preparations. The addition of catalase protected all systems, but catalase was only partially protective for 6-hydroxydopamine acting upon catecholamine uptake. The data show that 6-hydroxydopamine generates hydrogen peroxide and that hydrogen peroxide can damage the biogenic amine uptake systems. Part of the damage caused by the 6-hydroxydopamine that accumulates in the catecholamine nerve terminals in vivo may be attributed to the hydrogen peroxide.
Amines↗
An iron chelator is not required for reductive iron release from ferritin by radical generating systems.
Although a number of reducing systems can release iron from ferritin, there is debate as to whether the process additionally requires a chelator. We have studied ferritin iron release by microsomes, paraquat and NADPH, by dialuric acid and by hypoxanthine and xanthine oxidase, using ferrozine to complex the released iron. In each case, Fe2+ (ferrozine) formation was detectable when the ferrozine was added at the beginning of the 10 min reaction period, but not at the end. However, with catalase present, up to 0.7 times as much Fe2+ could be measured with ferrozine added at the end. Further Fe2+ could be recovered by adding ascorbate with the ferrozine. These results indicate that an iron chelator is not required for reductive iron release from ferritin. However, the released iron will not be detectable as Fe2+ unless it forms a complex that is resistant to oxidation by H2O2 or other oxidants.
Animals↗
Concerted action of reduced glutathione and superoxide dismutase in preventing redox cycling of dihydroxypyrimidines, and their role in antioxidant defence.
Dialuric Acid, the reduced form of the beta-cell toxin alloxan, and the related fava bean derivatives divicine and isouramil, autoxidize rapidly in neutral solution by a radical mechanism. GSH promotes redox cycling of each compound, with concomitant GSH oxidation and H2O2 production. With superoxide dismutase present, there is a lag period in which little oxidation occurs, followed by rapid oxidation. GSH extends this lag and decreases the subsequent rate of oxidation, so that with superoxide dismutase and a sufficient excess of GSH, coupled oxidation of GSH and each pyrimidine is almost completely suppressed. This mechanism may be a means whereby GSH in combination with superoxide dismutase protects against the cytotoxic effects of these reactive pyrimidines. Superoxide dismutase may also protect cells against oxidative stress in other situations where GSH acts as a radical scavenger, and we propose that the concerted action of GSH and superoxide dismutase constitutes an important antioxidant defence.
Antioxidants↗
Ferritin, lipid peroxidation and redox-cycling xenobiotics.
A number of xenobiotics are toxic because they redox cycle and generate free radicals. Interaction with iron, either to produce reactive species such as the hydroxyl radical, or to promote lipid peroxidation, is an important factor in this toxicity. A potential biological source of iron is ferritin. The cytotoxic pyrimidines, dialuric acid, divicine and isouramil, readily release iron from ferritin and promote ferritin-dependent lipid peroxidation. Superoxide dismutase and GSH, which maintain the pyrimidines in their reduced form, enhance both iron release and lipid peroxidation. Microsomes plus NADPH can reduce a number of iron complexes, although not ferritin. Reduction of Adriamycin, paraquat or various quinones to their radicals by the microsomes enhances reduction of the iron complexes, and in some cases, enables iron release from ferritin. Adriamycin stimulates iron-dependent lipid peroxidation of the microsomes. Ferritin can provide the iron, and peroxidation is most pronounced at low pO2. Complexing agents that suppress intracellular iron reduction and lipid peroxidation may protect against the toxicity of Adriamycin.
Animals↗
The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas.
Alloxan and streptozotocin are widely used to induce experimental diabetes in animals. The mechanism of their action in B cells of the pancreas has been intensively investigated and now is quite well understood. The cytotoxic action of both these diabetogenic agents is mediated by reactive oxygen species, however, the source of their generation is different in the case of alloxan and streptozotocin. Alloxan and the product of its reduction, dialuric acid, establish a redox cycle with the formation of superoxide radicals. These radicals undergo dismutation to hydrogen peroxide. Thereafter highly reactive hydroxyl radicals are formed by the Fenton reaction. The action of reactive oxygen species with a simultaneous massive increase in cytosolic calcium concentration causes rapid destruction of B cells. Streptozotocin enters the B cell via a glucose transporter (GLUT2) and causes alkylation of DNA. DNA damage induces activation of poly ADP-ribosylation, a process that is more important for the diabetogenicity of streptozotocin than DNA damage itself. Poly ADP-ribosylation leads to depletion of cellular NAD+ and ATP. Enhanced ATP dephosphorylation after streptozotocin treatment supplies a substrate for xanthine oxidase resulting in the formation of superoxide radicals. Consequently, hydrogen peroxide and hydroxyl radicals are also generated. Furthermore, streptozotocin liberates toxic amounts of nitric oxide that inhibits aconitase activity and participates in DNA damage. As a result of the streptozotocin action, B cells undergo the destruction by necrosis.
Alloxan↗
[Alloxan cytotoxicity: a new aspect of the problem].
The effect of alloxan on human intact erythrocytes (in the suspension and whole blood) was studied using an original potentiometric apparatus permitting continuous registration of the flow rate of reducing equivalents ('RE) through a plasmatic membrane. Acceleration of RE flow from erythrocytes due to the activation of hexosomonophosphate shunt (HMPS) was established. This effect was inhibited by P-chlormercury benzoate proving the involvement of the SH-groups of erythrocyte membrane proteins in the interaction of alloxan with erythrocytes. Oxygen consumption after adding alloxan was recorded by polarography that indicated the "start" of the alloxan----dialuric acid (A in equilibrium with DA) cycle. Enhanced RE production in erythrocytes may be caused by H2O2 and free radicals forming in this cycle. It was shown that HMPS activation i.e. transition to a more intense stationary regimen was maintained for a period exceeding the calculated period of alloxan "halt--life". This residual effect of alloxan "depleted" erythrocyte protective mechanisms that was expressed in a decrease in maximum capacity of HMPS of rat whole blood after receiving by the animals a diabetogenic dose of alloxan (150 mg/kg body mass). Thus a specific cumulation of the effect of alloxan expressed in the transition of the RE generation system to a more intense stationary regimen, was revealed. In investigating the mechanism of alloxan cytotoxicity one should take into account this peculiarity of alloxan along with the factor of affinity and conditions for initiation of the A in equilibrium with DA cycle.
Animals↗
Therapeutic efficacy of vitamin E and selenium in treating hemolytic anemia of owl monkeys (Aotus trivirgatus).
The roles played by vitamin E and by selenium in the anemia of owl monkeys were studied by injecting a mixture of the two antioxidants in 17 anemic monkeys. The result was an increase in mean hemoglobin concentration from 8.6 +/- 2.2 to 11.9 +/- 2.5 g/dl; in 1o of these monkeys with typically elevated numbers of circulating nucleated erythrocytes, the mean number of such cells per 10(2) leukocytes declined from 50.1 +/- 49.9 to 0.6 +/- 1.2. Injections of vitamin E alone in eight monkeys were less consistently effective; selenium administered alone to nine monkeys had no therapeutic effect. Neither anemic nor normal owl monkeys were deficient in serum vitamin E, plasma selenium, erythrocyte glutathione peroxidase or other related erythrocyte enzymes. Oral administration of vitamin E brought about no increase in serum levels of the vitamin, but hemolysis of erythrocytes in dialuric acid decreased from 18.6 +/- 15.8% to 1.0 +/- 0.03%. Selenium therapy brought about increased plasma selenium in two of three experiments, but glutathione peroxidase, which is selenium dependent, did not increase.
Administration, Oral↗