Damage to erythrocytes caused by the interaction of nitrite-ions with hemoglobin

The formation of two hemoglobin forms (methemoglobin and nitrite methemoglobin) in native human erythrocytes in the presence of sodium nitrite in suspension was shown. In normal erythrocytes, the interaction of intracellular oxyhemoglobin with nitrite ions results in the formation of methemoglobin, whereas in metabolically exhausted erythrocytes, this leads predominantly to the formation of nitrite methemoglobin. The nitrite methemoglobin reacts with hydrogen peroxide to form reactive intermediates (e.g. peroxynitrous acid) and the products of hemoglobin destruction. During the storage of erythrocyte suspensions containing methemoglobin and modified nitrite methemoglobin, differences in the forms of erythrocytes and the degree of their hemolysis were revealed. It is assumed that the formation of methemoglobin leads to the destruction of erythrocytes.     

A nonenzymatic method for determination of hydrogen peroxide and organic peroxides

Reduction of hydrogen peroxide and organic peroxides (t-butyl hydroperoxide and linoleic acid hydroperoxide) was achieved with homovanillic acid as hydrogen donor in the presence of the triethylenetetramine-Fe3+ complex. By the catalytic action of this complex, homovanillic acid is oxidized to its fluorescent dimer. Based on this reaction a fluorometric method for the measurement of the hydroperoxides mentioned above is described. The method can be extended to the determination of substrate-enzyme systems that produce hydrogen peroxide, e.g., glucose-glucose oxidase. The method allows the determination of substances such as hydrogen peroxide and t-butyl hydroperoxide with an accuracy and precision of less than 3%. Glucose can be determined with similar precision and an accuracy of 4.7%.     

Oxidation of iron-nitrosyl-hemoglobin by dehydroascorbic acid releases nitric oxide to form nitrite in human erythrocytes

The reaction of deoxyhemoglobin with nitric oxide (NO) or nitrite ions (NO 2 (-)) produces iron-nitrosyl-hemoglobin (HbNO) in contrast to the reaction with oxyhemoglobin, which produces methemoglobin and nitrate (NO 3 (-)). HbNO has not been associated with the known bioactivities of NO. We hypothesized that HbNO in erythrocytes could be an important source of bioactive NO/nitrite if its oxidation was coupled to the ascorbic acid (ASC) cycle. Studied by absorption and electron paramagnetic resonance (EPR) spectroscopy, DHA oxidized HbNO to methemoglobin and liberated NO from HbNO as determined by chemiluminescence. Both DHA and ascorbate free radical (AFR), the intermediate between ASC and DHA, enhanced NO oxidation to nitrite, but not nitrate; nor did either oxidize nitrite to nitrate. DHA increased the basal levels of nitrite in erythrocytes, while the reactions of nitrite with hemoglobin are slow. In erythrocytes loaded with HbNO, HbNO disappeared after DHA addition, and the AFR signal was detected by EPR. We suggest that the ASC-AFR-DHA cycle may be coupled to that of HbNO-nitrite and provide a mechanism for the endocrine transport of NO via hemoglobin within erythrocytes, resulting in the production of intracellular nitrite. Additionally, intracellular nitrite and nitrate seem to be largely generated by independent pathways within the erythrocyte. These data provide a physiologically robust mechanism for erythrocytic transport of NO bioactivity allowing for hormone-like properties.     

Unsaturated fatty acid-dependent oxidation of epinephrine in homogenates of the gorgonian, Pseudoplexaura porosa

A novel epinephrine oxidation system in homogenates of the gorgonian Pseudoplexaura porosa was discovered. The enzymatic reaction required an unsaturated fatty acid and molecular oxygen or hydrogen peroxide. Diphenylisobenzofuran was also oxidized by Ps. porosa homogenates in the presence of an unsaturated fatty acid. Hydroxyl radical and superoxide anion did not appear to be involved in either of these oxidative reactions. The production of lipid hydroperoxides was not necessary for epinephrine oxidation and, with the exception of arachidonic acid, lipid hydroperoxide production did not occur. Evidence is presented for the involvement of singlet oxygen or a similar activated oxygen intermediate in the reactions, and a possible mechanism was proposed. The use of arachidonate-dependent epinephrine oxidation as a measure of prostaglandin synthetase activity is criticized.     

Characterization of vanadium bromoperoxidase from Macrocystis and Fucus: reactivity of vanadium bromoperoxidase toward acyl and alkyl peroxides and bromination of amines

Vanadium bromoperoxidase (V-BrPO) has been isolated and purified from the marine brown algae Fucus distichus and Macrocystis pyrifera. V-BrPO catalyzes the oxidation of bromide by hydrogen peroxide, resulting in the bromination of certain organic acceptors or the formation of dioxygen. V-BrPO from F. distichus and M. pyrifera have subunit molecular weights of 65,000 and 74,000, respectively, and specific activities of 1580 units/mg (pH 6.5) and 1730 units/mg (pH 6) for the bromination of monochlorodimedone, respectively. As isolated, the enzymes contain a substoichiometric vanadium/subunit ratio; the vanadium content and specific activity are increased by addition of vanadate. V-BrPO (F. distichus, M. pyrifera, and Ascophyllum nodosum) also catalyzes the oxidation of bromide using peracetic acid. In the absence of an organic acceptor, a mixture of oxidized bromine species (e.g., hypobromous acid, bromine, and tribromide) is formed. Bromamine derivatives are formed from the corresponding amines, while 5-bromocytosine is formed from cytosine. In all cases, the rate of the V-BrPO-catalyzed reaction is much faster than that of the uncatalyzed oxidation of bromide by peracetic acid, at pH 8.5, 1 mM bromide, and 2 mM peracetic acid. In contrast to hydrogen peroxide, V-BrPO does not catalyze formation of dioxygen from peracetic acid in either the presence or absence of bromide. V-BrPO also uses phenylperacetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid to catalyze the oxidation of bromide; dioxygen is not formed with these peracids. V-BrPO does not catalyze bromide oxidation or dioxygen formation with the alkyl peroxides ethyl hydroperoxide, tert-butyl hydroperoxide, and cuminyl hydroperoxide.     

Inhibition of arachidonic acid incorporation into erythrocyte phospholipids by peracetic acid and other peroxides. Role of arachidonoyl-CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase

To explore possible mechanisms of the arachidonic acid deficiency of the red blood cell membrane in alcoholics, we compared the effect of ethanol and its oxidized products, acetaldehyde and peracetic acid, with other peroxides on the accumulation of [14C]arachidonate into RBC membrane lipids in vitro. Incubation of erythrocytes with 50 mM ethanol or 3 mM acetaldehyde had no effect on arachidonate incorporation. Pretreatment of erythrocytes with 10 mM hydrogen peroxide, 0.1 mM cumene hydroperoxide or 0.1 mM t-butyl hydroperoxide had little effect on [14C]arachidonate incorporation in the absence of azide. However, pretreatment of cells with N-ethylmaleimide, 0.1 mM peracetic acid or performic acid, with or without azide, inhibited arachidonate incorporation into phospholipids but not neutral lipids. In chase experiments, peracetate also inhibited transfer of arachidonate from neutral lipids to phospholipids. To investigate a possible site of this inhibition of arachidonate transfer into phospholipids by percarboxylic acids, we assayed a repair enzyme, arachidonoyl CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase (EC 2.3.1.23). As in intact cells, phospholipid biosynthesis was inhibited more by N-ethylmalemide and peracetic acid than by hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. Peracetic acid was the only active inhibitor among ethanol and its oxidized products studied and may deserve further examination in ethanol toxicity.     

Hydroperoxide-dependent folic acid degradation by cytochrome c

Folic acid is degraded by cytochrome c in the presence of hydrogen peroxide/tert-butyl hydroperoxide at the C9-N10 bond. The degradation is increased with increasing temperature. When guanidine HCl or benzoate are included in the reaction medium, the amount of folic acid degradation is enhanced. Catalase, formate, and thiourea inhibited hydrogen peroxide-dependent folic acid degradation only, and not tert-butyl hydroperoxide dependent degradation. Cyanide and azide markedly inhibited both the hydroperoxide-dependent degradations. Superoxide dismutase, EDTA, ethanol, mannitol, and dimethyl sulfoxide did not inhibit the degradation. The mechanism of cytochrome c-catalyzed folic acid degradation is discussed.     

Proteolysis in human erythrocytes is triggered only by selected oxidative stressing agents

Human erythrocytes have been treated with different agents producing oxidative stress. Diamide, tetrathionate, chromate, cystamine and iodate preferentially influenced the cellular redox state by oxidation of free and protein thiol groups leaving the redox state of hemoglobin virtually unaffected. None of these compounds was able to stimulate the proteolysis. By contrast, phenylhydrazine, nitrite, hydrogen peroxide, ter-butylhydroperoxide, cumene hydroperoxide and copper-ascorbate caused a noticeable oxidation of hemoglobin to methemoglobin. These latter agents, except nitrite and copper-ascorbate, triggered proteolysis. Identical results have been obtained in a ghost-free hemolysate. The fraction containing the proteolytic activity was isolated from hemolysate and tested on native or oxidant-treated hemoglobin. The proteolysis was stimulated by all agents able to produce methemoglobin. It is concluded that proteolysis correlated to an unbalance of cellular redox state. The results obtained with isolated and recombined fractions suggests that increased proteolysis does not depend on the removal of the effect of protease(s) inhibitor(s). Since all agents stimulating proteolysis are able to generate free radicals, it seems that protein breakdown is triggered by the direct effect of these intermediates on proteins (mostly hemoglobin) without the involvement of radical species produced in the membranes by action of organic hydroperoxides. In addition, since nitrite and copper-ascorbate, which also oxidize hemoglobin by radical generation, are unable to stimulate proteolysis, it should be concluded that protein degradation induced by oxidative stress is a complex event induced only by specific agents.     

The role of superoxide in xanthine oxidase-induced autooxidation of linoleic acid

The effect of hydroxyperoxyoctadecadienoic acid, e.g. 13-hydroperoxy-cis,9,trans-11-octadecadienoic acid, on the autooxidation of linoleic acid induced by superoxide radical was examined in a system containing xanthine oxidase, acetaldehyde, and diethylenetriaminepentaacetic acid dissolved in an aqueous phosphate buffer containing 10% ethanol. The superoxide radical is required for autooxidation, as shown by essentially complete inhibition on the addition of superoxide dismutase. Pure linoleic acid was not readily oxidized, but the addition of lipid hydroperoxide markedly stimulated the autooxidation. Addition of 2.8 microM FeCl3 did not produce an increase in the rate of xanthine oxidase-induced autooxidation. Spontaneous autooxidation, a process slower than xanthine oxidase-induced autooxidation, was detectable on the time scale of these observations but was slower than the xanthine oxidase-induced autooxidation. Initiation of linoleic acid autooxidation is postulated to result from a reaction between superoxide and lipid hydroperoxide. The nature of this reaction is uncertain, but it does not appear to depend on iron catalysis.     

Inhibition by amines indicates involvement of nitrogen dioxide in autocatalytic oxidation of oxyhemoglobin by nitrite

Oxidation of oxyhemoglobin by nitrite is characterized by a lag period followed by an autocatalytic phase. The oxidation can be inhibited by the addition of morpholine, piperidine, triethanolamine or triethylamine (6 mM each). These amines are known to react with nitrogen dioxide to yield nitrosamine. Unexpectedly, aniline or aminopyrine (120 microM each) markedly inhibited the oxidation. These compounds, but not the other amines given above, inhibited the peroxide compound formation from methemoglobin and hydrogen peroxide. The results establish that, during the oxidation, the peroxide compound is generated and converts nitrite into nitrogen dioxide by its peroxidatic activity, resulting in an autocatalytic phase.     

Studies on hydroperoxide-dependent folic acid degradation by hemin

Hemin (ferric protoporphyrin IX chloride) in the presence of hydrogen peroxide or tert-butyl hydroperoxide was found to cleave folic acid at the C9-N10 bond. The ferrous form of hemin was not involved in hydroperoxide-dependent folic acid degradation, as indicated by the lack of inhibition by carbon monoxide. Molecular oxygen was not required for the degradation. GSH-Mn(II) or NAD(P)H in the presence of molecular oxygen did not support hemin-mediated folic acid degradation. The degradation increased as the temperature was elevated from 10 to 70 degrees C. Ascorbic acid and azide were potent inhibitors. Superoxide dismutase and hydroxyl radical quenchers, such as ethanol, mannitol, benzoate, and dimethyl sulfoxide did not inhibit the reaction. Catalase inhibited hydrogen peroxide-supported degradation but not the tert-butyl hydroperoxide-dependent one. Thiol compounds, such as thioglycolic acid, thiourea, glutathione, cysteine, and 2-mercaptoethanol, inhibited the hydrogen peroxide-dependent degradation but supported the tert-butyl hydroperoxide-mediated one. N5-formyl tetrahydrofolic acid, but not N10-formyl folic acid, was degraded by hemin in the presence of H2O2 or TBHP. The data obtained are suggestive of a mechanism similar to N-demethylation reactions catalyzed by cytochrome P-450 and some peroxidases.     

Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis

The metabolic disorder, alkaptonuria, is distinguished by elevated serum levels of 2,5-dihydroxyphenylacetic acid (homogentisic acid), pigmentation of cartilage and connective tissue and, ultimately, the development of inflammatory arthritis. Oxygen radical generation during homogentisic acid autoxidation was characterized in vitro to assess the likelihood that oxygen radicals act as molecular agents of alkaptonuric arthritis in vivo. For homogentisic acid autoxidized at physiological pH and above, yielding superoxide (O2-)2 and hydrogen peroxide (H2O2), the homogentisic acid autoxidation rate was oxygen dependent, proportional to homogentisic acid concentration, temperature dependent and pH dependent. Formation of the oxidized product, benzoquinoneacetic acid was inhibited by the reducing agents, NADH, reduced glutathione, and ascorbic acid and accelerated by SOD and manganese-pyrophosphate. Manganese stimulated autoxidation was suppressed by diethylenetriaminepentaacetic acid (DTPA). Homogentisic acid autoxidation stimulated a rapid cooxidation of ascorbic acid at pH 7.45. Hydrogen peroxide was among the products of cooxidation. The combination of homogentisic acid and Fe3+-EDTA stimulated hydroxyl radical (OH.) formation estimated by salicylate hydroxylation. Ferric iron was required for the reaction and Fe3+-EDTA was a better catalyst than either free Fe3+ or Fe3+-DTPA. SOD accelerated OH. production by homogentisic acid as did H2O2, and catalase reversed much of the stimulation by SOD. Catalase alone, and the hydroxyl radical scavengers, thiourea and sodium formate, suppressed salicylate hydroxylation. Homogentisic acid and Fe3+-EDTA also stimulated the degradation of hyaluronic acid, the chief viscous element of synovial fluid. Hyaluronic acid depolymerization was time dependent and proportional to the homogentisic acid concentration up to 100 microM. The level of degradation observed was comparable to that obtained with ascorbic acid at equivalent concentrations. The hydroxyl radical was an active intermediate in depolymerization. Thus, catalase and the hydroxyl radical scavengers, thiourea and dimethyl sulfoxide, almost completely suppressed the depolymerization reaction. The ability of homogentisic acid to generate O2-, H2O2 and OH. through autoxidation and the degradation of hyaluronic acid by homogentisic acid-mediated by OH. production suggests that oxygen radicals play a significant role in the etiology of alkaptonuric arthritis.     

Different effects of Triton X-100, deoxycholate, and fatty acids on the kinetics of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase

The effects of Triton X-100, deoxycholate, and fatty acids were studied on the two steps of the ping-pong reaction catalyzed by Se-dependent glutathione peroxidases. The study was carried out by analyzing the single progression curves where the specific glutathione oxidation was monitored using glutathione reductase and NADPH. While the "classic" glutathione peroxidase was inhibited only by Triton, the newly discovered "phospholipid hydroperoxide glutathione peroxidase" was inhibited by deoxycholate and by unsaturated fatty acids. The kinetic analysis showed that in the case of glutathione peroxidase only the interaction of the lipophilic peroxidic substrate was hampered by Triton, indicating that the enzyme is not active at the interface. Phospholipid hydroperoxide glutathione peroxidase activity measured with linoleic acid hydroperoxide as substrate, on the other hand, was not stimulated by the Triton concentrations which have been shown to stimulate the activity on phospholipid hydroperoxides. Furthermore a slight inhibition was apparent at high Triton concentrations and the effect could be attributed to a surface dilution of the substrate. Deoxycholate and unsaturated fatty acids were not inhibitory on glutathione peroxidase but inhibited both steps of the peroxidic reaction of phospholipid hydroperoxide glutathione peroxidase, in the presence of either amphiphilic or hydrophilic substrates. This inhibition pattern suggests an interaction of anionic detergents with the active site of this enzyme. These results are in agreement with the different roles played by these peroxidases in the control of lipid peroxide concentrations in the cells. While glutathione peroxidase reduces the peroxides in the water phase (mainly hydrogen peroxide), the new peroxidase reduces the amphyphilic peroxides, possibly at the water-lipid interface.     

Singlet oxygen production by bleomycin. A comparison with heme-containing compounds

Fe(III)-bleomycin catalyzes the decomposition of 13-hydroperoxylinoleic acid and of 15-hydroperoxyarachidonic acid to produce small quantities of singlet oxygen. No singlet oxygen is produced when hydrogen peroxide, ethyl hydroperoxide, cumene hydroperoxide, and t-butyl hydroperoxide are used as substrates. The heme-containing catalysts, methemoglobin and hematin, have identical hydroperoxide substrate requirements for singlet oxygen production. The hydroperoxide requirements for singlet oxygen production correlate with those reported by Dix et al. (Dix, T.A., Fontana, R., Panthani, A., and Marnett, L.J. (1985) J. Biol. Chem. 260, 5358-5365) for the production of peroxyl radicals in the hematin-catalyzed decomposition of hydroperoxides. The bimolecular reaction of peroxyl radicals is a plausible reaction mechanism for the singlet oxygen production in the systems studied.     

A new spectrophotometric assay for enzymes of purine metabolism. II. Determination of guanase activity.

A new method for the determination of guanase is described. Xanthine, the product of the guanase reaction, is oxidized by xanthine oxidase, forming uric acid and hydrogen peroxide. Hydrogen peroxide is further reduced to water by catalase in the presence of ethanol. The acetaldehyde formed in this reaction step is dehydrogenated NAD or NADP dependent by aldehyde dehydrogenase. The NADH or NADPH production is measured and utilized for the calculation of the guanase activity. The sensitivity of the method can be doubled by the addition of uricase, which oxidizes uric acid to permit the formation of another mole of hydrogen peroxide.     

t-Butyl hydroperoxide alters fatty acid incorporation into erythrocyte membrane phospholipid

Because the ability of cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be an important determinant of the ability of cells to tolerate oxidative stress, incorporation of exogenous fatty acid into phospholipid by human erythrocytes has been examined following exposure of the cells to t-butyl hydroperoxide. Exposure of human erythrocytes to t-butyl hydroperoxide (0.5-1.0 mM) results in oxidation of glutathione, formation of malonyldialdehyde, and oxidation of hemoglobin to methemoglobin. Under these conditions, incorporation of exogenous [9,10-3H]oleic acid into phosphatidylethanolamine is enhanced while incorporation of [9,10-3H]oleic acid into phosphatidylcholine is decreased. These effects of t-butyl hydroperoxide on [9,10-3H]oleic acid incorporation are not affected by dissipating transmembrane gradients for calcium and potassium. When malonyldialdehyde production is inhibited by addition of ascorbic acid, t-butyl hydroperoxide still decreases [9,10-3H]oleic acid incorporation into phosphatidylcholine but no stimulation of [9,10-3H]oleic acid incorporation into phosphatidylethanolamine occurs. In cells pre-treated with NaNO2 to convert hemoglobin to methemoglobin, t-butyl hydroperoxide reduces [9,10-3H]oleic acid incorporation into phosphatidylcholine by erythrocytes but does not stimulate [9,10-3H]oleic acid incorporation into phosphatidylethanolamine. Under these conditions oxidation of erythrocyte glutathione and formation of malonyldialdehyde still occur. These results indicate that membrane phospholipid fatty acid turnover is altered under conditions where peroxidation of membrane phospholipid fatty acids occurs and suggest that the oxidation state of hemoglobin influences this response.     

Albumin oxidation to diverse radicals by the peroxidase activity of Cu,Zn-superoxide dismutase in the presence of bicarbonate or nitrite: diffusible radicals produce cysteinyl and solvent-exposed and -unexposed tyrosyl radicals

The peroxidase activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) has been extensively studied in recent years due to its potential relationship to familial amyotrophic lateral sclerosis. The mechanism by which Cu,Zn-SOD/hydrogen peroxide/bicarbonate is able to oxidize substrates has been proposed to be dependent on an oxidant whose nature, diffusible carbonate radical anion or enzyme-bound peroxycarbonate, remains debatable. One possibility to distinguish these species is to examine whether protein targets are oxidized to protein radicals. Here, we used EPR methodologies to study bovine serum albumin (BSA) oxidation by Cu,Zn-SOD/hydrogen peroxide in the absence and presence of bicarbonate or nitrite. The results showed that BSA oxidation in the presence of bicarbonate or nitrite at pH 7.4 produced mainly solvent-exposed and -unexposed BSA-tyrosyl radicals, respectively. Production of the latter was shown to be preceded by BSA-cysteinyl radical formation. The results also showed that hydrogen peroxide/bicarbonate extensively oxidized BSA-cysteine to the corresponding sulfenic acid even in the absence of Cu,Zn-SOD. Thus, our studies support the idea that peroxycarbonate acts as a two-electron oxidant and may be an important biological mediator. Overall, the results prove the diffusible and radical nature of the oxidants produced during the peroxidase activity of Cu,Zn-SOD in the presence of bicarbonate or nitrite.     

Enzymatic reaction of beta-N-methylaminoalanine with L-amino acid oxidase

The reaction of beta-N-methylaminoalanine (BMAA) with L-amino acid oxidase (L-AAO) in the presence of catalase yields ammonia and beta-N-methylaminopyruvate, which was trapped as its 2,4-dinitrophenylhydrazone, as products. Incubation of BMAA with L-AAO in the presence of semicarbazide led to the formation of a semicarbazone, indicating intermediate iminium ion formation; when potassium cyanide (5 mM) was added, semicarbazone formation was blocked. The formation of beta-N-methylaminopyruvate was decreased by omission of catalase and was reduced in the presence of hydrogen peroxide (100 mM). These results indicate that BMAA is converted by L-AAO to the corresponding alpha-imino acid, which undergoes hydrolysis to beta-N-methylaminopyruvate. The alpha-keto acid is readily oxidized to N-methylglycine by hydrogen peroxide.     

Theoretical analysis of a site-specific chemiluminescence reaction and its application to quantitation of lipid hydroperoxides

A system was designed for chemiluminescent measurement of lipid hydroperoxides by their site-specific reaction in sodium dodecylsulfate micelles. Ferrous ion-induced decomposition of lipid hydroperoxides in the sodium dodecylsulfate micelles resulted in strong chemiluminescence of the Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (CLA). After addition of ferrous sulfate to the micelles containing lipid hydroperoxide and luciferin, the chemiluminescence intensity reached a maximum rapidly and then decreased. The sequence of this reaction was elucidated by theoretical analysis, which demonstrated that the maximum chemiluminescence intensity is proportional to the initial concentration of hydroperoxide. Good linear relationships were observed between the maximum counts of chemiluminescence and the amounts of hydroperoxides of linoleic acid, phosphatidylcholine, choresterol (5 alpha), cumene and tert-butyl and hydrogen peroxide. This chemiluminescence method was simple and sensitive enough to detect picomole levels of linoleic acid and phosphatidylcholine hydroperoxides.     

Reaction of carbohydrates with hydroperoxides : Part II. Oxidation of ketoses with the hydroperoxide anion

When treated with an excess of hydrogen peroxide under alkaline conditions, D-fructose and L-sorbose yield approximately four moles of formic acid and one mole of glycolic acid per mole of hexulose. This observation is rationalized by a reaction mechanism consisting of nucleophilic addition of a hydroperoxide anion to the carbonyl form of the ketose, followed by oxidative cleavage of the hydroperoxide adduct to glycolic acid and the next lower aldose. The aldose is subsequently degraded entirely to formic acid by the mechanism described in the preceding paper of this series. Oxidative cleavage of the hydroperoxide adduct of a hexulose could take place by rupture of either the C-1C-2 or the C-2C-3 bond. The reaction products show that, under the conditions used, the oxidation takes place almost entirely with cleavage of the C-2C-3 bond of the parent sugar. The optical rotations of the reaction mixtures approach zero as the reactions proceed, and there is no indication of accumulation of optically active intermediates.In buffered solutions, the rate of reaction increases linearly with the increase in the concentration of alkali peroxide. With a constant amount of hydrogen peroxide and increasing amounts of alkali, the rate increases to a maximum corresponding to approximately one equivalent of alkali per mole of peroxide. The rate then decreases slightly as the pH is increased from 12.3 to 13.9. Lack of a rate increase in this range suggests a free-radical, rather than a base-catalyzed, mechanism for the oxidative cleavage of the adduct.