Superoxide, hydrogen peroxide and singlet oxygen in hematoporphyrin derivative-cysteine, -NADH and -light systems

Hematoporphyrin derivative and light in the presence of cysteine or glutathione were found to convert oxygen to superoxide and hydrogen peroxide at pH less than approx. 6.5, while at pH greater than 6.5 no superoxide or hydrogen peroxide production was observed. However, at pH values greater than 6.5 the rate of oxygen consumption increased. This rate paralleled the acid dissociation curve of the cysteine thiol group and is consistent with the chemical quenching of 1O2 by cysteine. The superoxide and hydrogen peroxide formation observed below pH 6.5 appeared not to be related to the singlet oxygen production of hematoporphyrin derivative. In addition, superoxide and hydrogen peroxide production was observed with hematoporphyrin derivative and light in the presence of NADH, both above and below pH 6.5. Direct detection of singlet oxygen luminescence at 1268 nm in the hematoporphyrin derivative-light system (2H2O as solvent) revealed an apparent linear increase in the singlet oxygen emission intensity as the p2H was raised from 7.0 to 10.0. Azide efficiently quenched this observed emission. In addition, at p2H 7.4, 1 mM cysteine resulted in a 40% reduction of the singlet oxygen luminescence, while at p2H 9.4 the signal was quenched by over 95% (under the experimental conditions employed). In total, we interpret these results as consistent with the chemical quenching of 1O2 by the ionized thiol group of cysteine.     

Pyridine nucleotide-dependent generation of hydrogen peroxide by a particulate fraction from human neutrophils

The catalytic oxidation of [14C]-formate to 14CO2 was adapted to measure H2O2 formation in cellfree system. Standard curves employing glucose-glucose oxidase and xanthine-xanthine oxidase demonstrated linearity between 14CO2 evolution and enzyme concentration. A particulate fraction from human neutrophils was capable of oxidizing [14C]-formate; this reaction was dependent upon the presence of catalase, reduced pyridine nucleotide, and cellular material. Reaction increased with time of incubation and protein concentration, although not in a strictly linear fashion. The pH optimum was approximately 5.5 NADPH was a significantly better substrate than NADH, although both were capable of generating H2O2. The particulate fraction derived from phagocytizing cells was more active than a corresponding fraction from resting cells with either substrate. H2O2 production was abnormal in particulate fractions derived from 2 patients with chronic granulomatous disease. H2O2 production was markedly inhibited by superoxide dismutase or cytochrome c (scavengers of superoxide anion) but not by scavengers of singlet oxygen or hydroxyl radical. Reaction was greatly stimulated by the addition of manganous ion. These results are consistent with the hypothesis that the respiratory burst in human neutrophils is initiated by an oxidase that can utilize either NADPH or NADH but exhibits a marked preference for the former. Further, the inhibitor studies strongly support a mechanism involving an initial enzymatic reaction followed by a self-sustaining free radical reaction involving superoxide anion.     

Increased phospholipid methylation in the myocardium of alcoholic rats

NAD(P)H is known to be oxidized by singlet molecular oxygen, perhydroxyl radical, and hydroxyl radical. In marked contrast to these reactive oxygen species, NAD(P)H is stable in the presence of micromolar concentrations of H2O2. The experiments herein demonstrate that NADPH is rapidly oxidized by H2O2 in the presence of a heme-peptide. The oxidation product is enzymatically active NADP+. In the absence of NADPH, the heme-peptide undergoes rapid degradation via reaction with H2O2. In the presence of NADPH, the reduced nucleotide is oxidized to NADP and the heme-peptide is partially protected from oxidation. It is suggested that under certain conditions the reduced nucleotides may contribute to the protection of intracellular heme moieties from degradation engendered by endogenous or exogenous H2O2.     

NADPH oxidation catalyzed by the peroxidase/H2O2 system. Guaiacol-mediated and scopoletin-mediated oxidation of NADPH to NADPH+

We have examined the respective roles played by guaiacol and scopoletin in NADPH oxidation catalyzed by the peroxidase/H2O2 system. It was shown that NADPH was not oxidized by either the horseradish or lactoperoxidase/H2O2 systems alone; oxidation occurred immediately after the addition of guaiacol or scopoletin. In both cases, the oxidation product was enzymatically active NADP+. Differences were observed in the NADPH oxidation mechanism depending on whether guaiacol or scopoletin was the mediator molecule. In guaiacol-mediated NADPH oxidation, the stoichiometry between H2O2 and oxidized NADPH was about 1; superoxide dismutase did not affect the oxidation rate. In scopoletin-mediated oxidation, the stoichiometry was much higher (1:14 in the present experiments); superoxide dismutase considerably increased the oxidation rate. It is concluded that catalysis of NADPH oxidation by the horse radish peroxidase/H2O2 system requires the presence of a mediator molecule. The NADPH oxidation mechanism depends on the intermediary oxidation state of this molecule.     

Studies on the mechanism of NADPH oxidation by the granule fraction isolated from human resting polymorphonuclear blood cells.

Various factor affecting NADPH-oxidation by resting human leucocyte granules (LG) at acid pH, have been investigated. It was found that: 1) oxidation of NADPH by LG was increasingly inhibited by increased cyanide concentrations in the medium and was abolished by 4 mM cyanide. 2) with or without cyanide in the incubation medium, LG omitted, Mn++ in the presence of NADPH induced superoxide anion (O- WITH 2) production, as evidenced by oxygen consumption and H2O2 production, which were abolished (in the absence of cyanide) by cytochrome C (a potent O- with 2 scavenger). 3) Both NADPH oxidation in the presence of 2 mM cyanide (cyanide-resistant) and in its absence (cyanide-sensitive) by LG occurred only in the presence of Mn++, and both were inhibited by superoxide dismutase. 4) Cyanide-resistant NADPH oxidation by LG generated H2O2, was inhibited by H2O2 and was not modified by "active" catalase. The ratio of cyanide-resistant NADPH oxidation/O2 uptake was 1 up to 1.25 mM NADPH, and increased above this concentration. 5) Cyanide-sensitive NADPH oxidation was inhibited by catalase and increased upon addition of H2O2. The ratio of cyanide-sensitive NADPH oxidation/O2 uptake was 2. It was concluded that after initiation by O - with 2, produced independently of LG, two sequential types of LG dependent NADPH oxidations occur. First, an O - with 2-dependent protein mediated NADPH oxidation (cyanide-resistant) which generates H2O2 and O - with 2 occurs. Second, NADPH peroxidation (cyanide-sensitive) which utilizes H2O2 takes place.     

The role of superoxide and singlet oxygen in lipid peroxidation promoted by xanthine oxidase

The peroxidative oxidation of extracted rat liver microsomal lipid, assayed as malondialdehyde production, can be promoted by milk xanthine oxidase in the presence of 0.2 mM FeCl₃ and 0.1 mM EDTA. The reaction is inhibited by the superoxide dismutase activity of erythrocuprein. The reaction is also inhibited by 1,3-diphenylisobenzofuran, which reacts with singlet oxygen to yield dibenzoylbenzene. During inhibition of the lipid peroxidation reaction by 1,3-diphenylisobenzofuran, o-dibenzoylbenzene was produced. The rate of superoxide production by xanthine oxidase was not affected by 1,3-diphenylisobenzofuran. Lipid peroxidation promoted by ascorbic acid is not inhibited by either erythrocuprein or 1,3-diphenylisobenzofuran. Therefore it is suggested that the peroxidative oxidation of unsaturated lipid promoted by xanthine oxidase involves the formation of singlet oxygen from superoxide, and the singlet oxygen reacts with the lipid to form fatty acid hydroperoxides.     

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.     

Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes.

Glycerol can be oxidized by rat liver microsomes to formaldehyde in a reaction that requires the production of reactive oxygen intermediates. Studies with inhibitors, antibodies, and reconstituted systems with purified cytochrome P4502E1 were carried out to evaluate whether P450 was required for glycerol oxidation. A purified system containing phospholipid, NADPH-cytochrome P450 reductase, P4502E1, and NADPH oxidized glycerol to formaldehyde. Formaldehyde production was dependent on NADPH, reductase, and P450, but not phospholipid. Formaldehyde production was inhibited by substrates and ligands for P4502E1, as well as by anti-pyrazole P4502E1 IgG. The oxidation of glycerol by the reconstituted system was sensitive to catalase, desferrioxamine, and EDTA but not to superoxide dismutase or mannitol, indicating a role for H2O2 plus non-heme iron, but not superoxide or hydroxyl radical in the overall glycerol oxidation pathway. The requirement for reactive oxygen intermediates for glycerol oxidation is in contrast to the oxidation of typical substrates for P450. In microsomes from pyrazole-treated, but not phenobarbital-treated rats, glycerol oxidation was inhibited by anti-pyrazole P450 IgG, anti-hamster ethanol-induced P450 IgG, and monoclonal antibody to ethanol-induced P450, although to a lesser extent than inhibition of dimethylnitrosamine oxidation. Anti-rabbit P4503a IgG did not inhibit glycerol oxidation at concentrations that inhibited oxidation of dimethylnitrosamine. Inhibition of glycerol oxidation by antibodies and by aminotriazole and miconazole was closely associated with inhibition of H2O2 production. These results indicate that P450 is required for glycerol oxidation to formaldehyde; however, glycerol is not a direct substrate for oxidation to formaldehyde by P450 but is a substrate for an oxidant derived from interaction of iron with H2O2 generated by cytochrome P450.     

Activation by ATP of calcium-dependent NADPH-oxidase generating hydrogen peroxide in thyroid plasma membranes

An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.     

NADPH oxidation catalyzed by the peroxidase/H2O2 system. Iodide-mediated oxidation of NADPH to iodinated NADP

Oxidation of NADPH catalyzed by the peroxidase/H2O2 system is known to require the presence of mediating molecules. Using either lactoperoxidase or horseradish peroxidase, we demonstrated that in the peroxidase/H2O2 system, NADPH oxidation was mediated by iodide. The oxidation product was the iodinated NADP. This product was shown to possess spectral characteristics different from those of NADP+ and NADPH, since for iodinated NADP, increased absorbance was observed in the 280-nm region and was directly proportional to the rate of iodination. It is suggested that oxidation and iodination of NADPH proceed via a single reaction between the intermediary iodide oxidation species and NADPH. Experiments with different molecules of NADPH analogues indicated that iodination occurred in the nicotinamide part of the NADPH molecule.     

Role of cytosolic superoxide dismutase as a stimulator in anthranilamide hydroxylation by a microsomal monooxygenase system in rat liver

We have isolated a protein factor from rat liver which stimulates anthranilamide hydroxylation by the microsomes in the presence of NADPH and oxygen and showed this factor to contain Cu and Zn and to have superoxide dismutase activity [Biochim. Biophys. Acta 365, 148-157 (1974)]. In the present study, this protein factor was confirmed to be a superoxide dismutase (SOD) by comparison of the recovery of SOD activity with that of anthranilamide hydroxylation-stimulating activity at each step of its purification, by inhibition of SOD activity with NaCN and hydrogen peroxide (H2O2), and by recovery of the SOD activity of the protein factor after reconstitution with Cu2+ and/or Zn2+. At a given SOD activity level, there was no difference among the rat liver SOD, Cu,Zn-SOD from bovine erythrocytes, and Mn-SOD from Serratia marcescens in their ability to stimulate anthranilamide hydroxylation not only by rat liver microsomes, but also by the reconstituted cytochrome P-450-containing monooxygenase system. Rat liver SOD stimulated anthranilamide hydroxylation by the reconstituted system in proportion to its amount below a protein concentration of 1 microgram/ml. In anthranilamide hydroxylation by the reconstituted system without SOD, only a slight hydroxylase activity was found at the initial stage of the reaction and a marked increase in the amounts of NADPH oxidized and H2O2 formed was observed after a lag time. In the presence of rat liver SOD, however, the hydroxylase activity was markedly and continuously increased almost proportionally to reaction time with a concomitant decrease in the amounts of NADPH oxidized and H2O2 formed. In addition, a trace of 3-OH anthranilamide, one of the products, not only stimulated NADPH-dependent H2O2 formation in the reconstituted system, but also inhibited the apparent reduction of cytochrome P-450 by NADPH in the reconstituted system. These effects of 3-OH anthranilamide were diminished by rat liver SOD. When a trace of 3-OH anthranilamide were added to a system composed of NADPH-cytochrome c (P-450) reductase and NADPH, H2O2 formation and NADPH oxidation were markedly stimulated. However, on addition of 3-OH anthranilamide to the system containing rat liver SOD, no stimulation on either H2O2 formation or NADPH oxidation was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytochrome c enhancement of singlet molecular oxygen production by the NADPH-dependent adrenodoxin reductase-adrenodoxin system: the role of singlet oxygen in damaging adrenal mitochondrial membranes

In the presence of NADPH, cytochrome c stimulates approximately a 200-fold increase in the production of singlet oxygen by the bovine adrenodoxin reductase-adrenodoxin system. The formation of singlet oxygen, which was monitored by the attending chemiluminescence, was markedly inhibited by the addition of superoxide dismutase or 1,4-diazabicyclo[2.2.2]octane. The adrenal system, in the presence of cytochrome c, peroxidized adrenal mitochondrial lipids, as indicated by the formation of malondialdehyde. This oxidation is also inhibited by the addition of dismutase and 1,4-diazabicyclo[2.2.2]octane.     

On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction

This laboratory has recently reported that, in a reconstituted enzyme system containing alcohol-induced isozyme 3a of liver microsomal cytochrome P-450, the sum of acetaldehyde generated by the monooxygenation of ethanol and of hydrogen peroxide produced by the NADPH oxidase activity is inadequate to account for the O2 and NADPH consumed. Studies on the stoichiometry have revealed the occurrence of an additional reaction involving an overall 4-electron transfer to molecular oxygen which is presumed to yield water: O2 + 2 NADPH + 2H+----2 H2O + 2 NADP+. The occurrence of a peroxidase reaction in which free H2O2 is reduced to water by NADPH was ruled out. When the 4-electron oxidase activity is taken into account, measurements of NADPH oxidation and O2 consumption are in accord with the amounts of products formed in the presence of various P-450 isozymes, either in the absence or presence of typical substrates, including those which undergo hydroxylation, N- or O-demethylation, or oxidation of hydroxymethyl to aldehyde groups. Of the substrates examined, some had no effect on the oxidase reaction yielding hydrogen peroxide or the 4-electron oxidase reaction, some were inhibitory, and some were stimulatory, but the same substrate did not necessarily have the same effect on the two reactions.     

Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases

The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH:cytochrome b5 (EC 1.6.2.2) and NADPH:cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD+ and NADP+, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O2- than O2-2. We suggest that a ferroheme-O2 and various heme-oxy radical complexes (mainly ferroheme-O-2 complex) play a crucial role in NAD(P)H oxidation.     

Singlet oxygen formation by a peroxidase, H2O2 and halide system.

Evidence for singlet oxygen formation has been obtained for the lactoperoxidase, H2O2 and bromide system by monitoring 2,3-diphenylfuran and diphenylisobenzofuran oxidation, O2 evolution, and chemiluminescence. This could provide an explanation for the cytotoxic and microbicidal activity of peroxidases and polymorphonuclear leukocytes. Evidence for singlet oxygen formation included the following. (a) Chemiluminescence accompanying the enzymic reaction was doubled in a deuterated buffer and inhibited by singlet oxygen traps. (b) The singlet oxygen traps, diphenylfuran and diphenylisobenzofuran, were oxidized to their known singlet oxygen oxidation products in the presence of lactoperoxidase, hydrogen peroxide and bromide. (c) The rate of oxidation of diphenylfuran and diphenylisobenzofuran was inhibited when monitored in the presence of known singlet oxygen traps or quenchers. (d) Oxygen evolution from the enzymic reaction was inhibited by singlet oxygen traps but not by singlet oxygen quenchers. (e) The traps or quenchers which were effective inhibitors in the experiments above did not inhibit peroxidase activity, were not competitive peroxidase substrates and did not react with the hypobromite intermediate since they did not inhibit hydrogen peroxide consumption by the enzyme. Using these criteria, various biological molecules were tested for their reactivity with singlet oxygen. Furthermore, by studying their effect on oxygen release by the enzymic reaction, it could be ascertained whether they were acting as singlet oxygen traps or quenchers.     

Reduction of 2,4,6-trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer

Glutathione reductase has been found to catalyze an NAD(P)H-dependent electron transfer to 2,4,6-trinitrobenzenesulfonate (TNBS). In the presence of oxygen TNBS is not consumed in the reaction, but is rapidly reoxidized with concomitant production of hydrogen peroxide. Cytochrome c can replace oxygen as the final electron acceptor, indicating that a one-electron transfer takes place. The rate is slightly higher in the absence than in the presence of oxygen, ruling out superoxide anion as an obligatory intermediate in cytochrome c reduction. In the absence of oxygen (or cytochrome c), TNBS limits the reaction and accepts a total of four electrons. The TNBS-dependent NADPH (or NADH) oxidation is markedly stimulated by NADP+, and to a smaller extent also by NAD+. The TNBS-dependent reactions are inhibited by excess of NADPH but not by NADH. The kinetics of these reactions are consistent with a branching reaction mechanism in which a pathway including a ternary complex between the two-electron reduced enzyme and NADP+ has the highest turnover. NADPH-dependent reductions of ferricyanide or 2,6-dichloroindophenol catalyzed by glutathione reductase are also markedly influenced by NADP+. Evidently NADP+ facilitates a shift of the catalyzed reaction from the normal two-electron reduction of glutathione disulfide to a more unspecific one-electron reduction of other acceptors. Spectral as well as kinetic data suggest that the rate of radical formation limits the reactions with the artificial electron acceptors and that NADP+ promotes this rate-limiting step.     

Chemiluminescence probe with Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, for estimating the ability of human granulocytes to generate O2-

The Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), in Hanks' balanced salt solution, emitted a weak luminescence which was not affected by superoxide dismutase or catalase and was not augmented by resting human granulocytes. In contrast, activated granulocytes caused a dramatic increase in the luminescence of CLA. The light emission by CLA in the presence of activated granulocytes was inhibited by superoxide dismutase, but not by catalase or benzoate. Azide at 0.5 mM did not inhibit light emission significantly. These results indicate that O2-, rather than H2O2, HO., singlet oxygen, or HOCl, was the agent responsible for eliciting the chemiluminescence of CLA. Moreover, the intensity of light emission by CLA correlated with the rate of production of O2- either by activated neutrophils or by the xanthine oxidase reaction.     

NADH-dependent microsomal interaction with ferric complexes and production of reactive oxygen intermediates

The interaction of NADPH with ferric complexes to catalyze microsomal generation of reactive oxygen intermediates has been well studied. Experiments were carried out to characterize the ability of NADH to interact with various ferric chelates to promote microsomal lipid peroxidation and generation of .OH-like species. In the presence of NADH and iron, microsomes produced .OH as assessed by the oxidation of a variety of .OH scavenging agents. Rates of NADH-dependent .OH production were 50 to 80% those of the NADPH-catalyzed reaction. The oxidation of dimethyl sulfoxide or t-butyl alcohol was inhibited by catalase and competitive .OH scavengers but not by superoxide dismutase or carbon monoxide. NADH-dependent .OH production was effectively catalyzed by ferric-EDTA and ferric-diethylenetriaminepentaacetic acid (DTPA), whereas ferric-ATP and ferric-citrate were poor catalysts. All these ferric chelates were reduced by microsomes in the presence of NADH (and NADPH). H2O2 was produced in the presence of NADH in a reaction stimulated by the addition of ferric-EDTA, consistent with the increase in .OH production. The latter appeared to be limited by the rate of H2O2 generation rather than the rate of reduction of the ferric chelate. NADH-dependent lipid peroxidation was much lower than the NADPH-catalyzed reaction and showed an opposite response to catalysis by ferric complexes compared to .OH generation as production of thiobarbituric acid-reactive material was increased with ferric-ATP and -citrate, but not with ferric-EDTA or- DTPA, and was not affected by catalase, SOD, or .OH scavengers. These results indicate that NADH can support microsomal reduction of ferric chelates, with the subsequent production of .OH-like species and peroxidation of lipids. The pattern of response of the NADH-dependent reactions with respect to catalytic effectiveness of ferric chelates and sensitivity to radical scavengers is similar to that found with NADPH. Many of the metabolic actions of ethanol have been ascribed to production of NADH as a consequence of oxidation by alcohol dehydrogenase. Since the cytosol normally maintains a highly oxidized NAD+/NADH redox ratio, it is interesting to speculate that increased availability of NADH from the oxidation of ethanol may support microsomal reduction of iron complexes, with the subsequent generation of reactive oxygen intermediates.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes: evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 X g particulate fraction of the postnuclear supernatant obtained frm guinea-pig phagocytosing poymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O2- generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH. 2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1:1. 3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O2- generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation. 4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O2- formed during the oxidation of NADPH. 5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reactin at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH. 6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH. 7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible. 8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase. 9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O2; an enzymatic, apparently non-univalent, O2 reduction and a non-enzymatic chain reaction. 10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O2 is optimal at pH 7.0 with 1 mM NADPH.