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In addition to their role as oxygen transporters, red blood cells (RBCs) contribute to cardiovascular homeostasis by regulating nitric oxide (NO) metabolism via interaction of hemoglobin (Hb) with nitrite and NO itself. RBCs were proposed to also participate in sulfide metabolism. Although Hb is known to react with sulfide, sulfide metabolism by intact RBCs has not been characterized so far. Therefore we explored the role of Hb in sulfide metabolism in intact human RBCs. We find that upon exposure of washed RBCs to sulfide, no changes in oxy/deoxyhemoglobin (oxy/deoxyHb) are observed by UV-vis and EPR spectroscopy. However, sulfide reacts with methemoglobin (metHb), forming a methemoglobin-sulfide (metHb-SH) complex. Moreover, while metHb-SH is stable in cell-free systems even in the presence of biologically relevant thiols, it gradually decomposes to produce oxyHb, inorganic polysulfides and thiosulfate in intact cells, as detected by EPR and mass spectrometry. Taken together, our results demonstrate that under physiological conditions RBCs are able to metabolize sulfide via intermediate formation of a metHb-SH complex, which subsequently decomposes to oxyHb. We speculate that decomposition of metHb-SH is preceded by an inner-sphere electron transfer, forming reduced Hb (which binds oxygen to form oxyHb) and thiyl radical (a process we here define as "reductive sulfhydration"), which upon release, gives rise to the oxidized products, thiosulfate and polysulfides. Thus, not only is metHb an efficient scavenger and regulator of sulfide in blood, intracellular sulfide itself may play a role in keeping Hb in the reduced oxygen-binding form and, therefore, be involved in RBC physiology and function.

Metamizole is an analgesic and antipyretic , but can cause neutropenia and agranulocytosis . We investigated the toxicity of the metabolites N-methyl-4-aminoantipyrine (MAA), 4-aminoantipyrine (AA), N-formyl-4-aminoantipyrine (FAA) and N-acetyl-4-aminoantipyrine (AAA) on neutrophil granulocytes and on HL60 cells (granulocyte precursor cell line). MAA, FAA, AA, and AAA (up to 100 µM) alone were not toxic for HL60 cells or granulocytes. In the presence of the myeloperoxidase substrate H2O2, MAA reduced cytotoxicity for HL60 cells at low concentrations (

Poor glycaemic control in diabetes and a combination of oxidative, metabolic, and carbonyl stresses are thought to lead to widespread non-enzymatic glycation and eventually to diabetic complications. Diabetic tissues can suffer both restriction in their supply of reducing power and excessive demand for reducing power. This contributes to compromised antioxidant status, particularly in the essential glutathione maintenance system. To study and ultimately correct deficiencies in diabetic glutathione maintenance, an experimental model would be desirable, which would provide in vitro a rapid, convenient, and dynamic reflection of the performance of diabetic GSH antioxidant capacity compared with that of non-diabetics. Xenobiotic-mediated in vitro methaemoglobin formation in erythrocytes drawn from diabetic volunteers is significantly lower than that in erythrocytes of non-diabetics. Aromatic hydroxylamine-mediated methaemoglobin formation is GSH-dependent and is indicative of the ability of an erythrocyte to maintain GSH levels during rapid thiol consumption. Although nitrite forms methaemoglobin through a complex GSH-independent pathway, it also reveals deficiencies in diabetic detoxification and antioxidant performance compared with non-diabetics. Together with efficient glycaemic monitoring, future therapy of diabetes may include trials of different antiglycation agents and antioxidant combinations. Equalization in vitro of diabetic methaemoglobin generation with that of age/sex-matched non-diabetic subjects might provide an early indication of diabetic antioxidant status improvement in these studies.

The action of carvedilol, a vasodilating, beta-adrenoceptor blocking agent, against lipid peroxidation has been the subject of many studies, but the results reported thus far are contradictory. In an attempt to define the antioxidant mechanism of carvedilol against lipid peroxidation, the dynamics of the action of carvedilol were studied in several oxidation systems. We investigated the reactivity of carvedilol toward radicals and its inhibitory effect on lipid peroxidation induced by several kinds of initiating species such as azo compounds and metal ions in solution, micelles, membranes, and low-density lipoprotein. Carvedilol exerted poor reactivity toward phenoxyl, alkoxyl, and peroxyl radicals in acetonitrile solution nor did it show an appreciable antioxidant effect against either the peroxyl radical-induced oxidation of methyl linoleate in acetonitrile or against phosphatidylcholine liposomal membranes in aqueous suspension. Carvedilol completely inhibited the ferric ion-induced oxidation of methyl linoleate micelles by sequestering ferric ions, but not by reducing hydroperoxide. It was shown that carvedilol enhanced the oxidation of micelles induced by either methemoglobin or peroxyl radical. Carvedilol, which was added exogenously, did not suppress the oxidation of isolated low-density lipoprotein induced by peroxyl radical or cupric ion. These results show that carvedilol does not act as a radical-scavenging antioxidant, but that it does act most efficiently as an antioxidant against ferric ion-induced oxidation by sequestering ferric ion.

The goal of the present work was to determine whether nitric oxide (NO) released from different donors (NONOates and nitrosothiols) can act as a protective antioxidant against oxidative stress and cytotoxicity induced by extracellular hemoglobin/tert-butyl hydroperoxide (Hb/tert-BuOOH) in vascular smooth muscle cells (VSMCs). No changes in phospholipid composition were found in VSMCs incubated with oxyhemoglobin (oxyHb)/tert-BuOOH. Using our newly developed HPLC-fluorescence technique for measurement of site-specific oxidative stress in membrane phospholipids, we produced VSMCs in which endogenous phospholipids were metabolically labeled with an oxidation-sensitive fluorescent fatty acid, cis-parinaric acid. In these cells, we were able to reliably quantitate oxidative stress in major phospholipid classes-phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol-induced by tert-BuOOH in the presence of oxyHb or methemoglobin (metHb). The oxidative stress was accompanied by cytotoxic effects of oxyHb/tert-BuOOH and metHb/tert-BuOOH on VSMCs. We further found that an NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen 1-ium-1,2-diolate (PAPANONO), but not nitrosothiols, protected VSMCs against oxidative stress and cytotoxicity induced by Hb/tert-BuOOH. The protective effect of PAPANONO was most likely due to its ability to form NO-heme Hb (detectable by low temperature EPR spectroscopy and visible spectrophotometry). These findings are important for further understanding the physiological antioxidant role of NO against oxidative stress induced by hemoproteins as well as for pathological hypertensive events induced by extracellular Hb via NO depletion.

The adenylyl cyclase activity of homogenates of striatal tissue from rat brain has been used as a model to test the hypothesis that the products of the reaction of polyphenols with ferric iron compounds are toxic. Dopamine (DA), at levels that stimulate adenylyl cyclase, inhibited the activity in the presence of 2 mol of potassium ferricyanide (FC), methemoglobin or ferricytochrome c per mol of DA. Combinations of potassium ferrocyanide and DA were not inhibitory. Neither pyrocatechol nor hydroquinone stimulated the activity, but these polyphenols were inhibitory in the presence of FC. Tyramine, phosphorylated DA or phosphorylated pyrocatechol had no effect on the activity of the enzyme in the presence or absence of FC. Forskolin was unable to stimulate the adenylyl cyclase once the latter was inhibited by DA plus FC, and dithiothreitol could not reverse inhibition by DA plus FC. Incubation of DA with FC, in the absence of the homogenate, resulted in substances that were not inhibitory. These findings suggest that the polyphenols plus FC react to yield substances that inhibit the adenylyl cyclase by affecting the catalytic unit of the enzyme complex.

We have used an in vitro two-compartment model, to investigate the ability of dapsone, formed by erythrocyte-mediated detoxification of its hydroxylamine metabolite, to escape the cells and cross a semi-permeable membrane into both plasma and other erythrocytes. Both diethyl dithiocarbamate (DDC) treated and untreated erythrocytes were incubated with dapsone hydroxylamine and dialysed against either fresh cells or plasma. Methaemoglobin was predominantly detectable in compartment A although the presence of low levels of methaemoglobin in compartment B indicated that the hydroxylamine itself had crossed the membrane. In contrast to methaemoglobin disposition, recovery of dapsone was higher (P

The effects of the di-catechols rooperol [(E)-1,5-bis(3',4'- dihydroxyphenyl)pent-4-en-1-yne; P2] and nordihydroguaiaretic acid (NDGA) on oxidative systems in the human blood were investigated. P2 and NDGA gave comparable results in the inhibition of leukotriene synthesis in the polymorphonuclear leukocyte and prostaglandin synthesis in platelet microsomes. The oxidation states of myeloperoxidase in the presence of H2O2 were also similarly affected by both P2 and NDGA. In these systems, the 4'4'-beta-D-diglucopyranoside of rooperol, hypoxoside [(E)-1,5-bis(4' beta-D-glucopyranosyloxy-3'- hydroxyphenyl) pent-4-en-1-yne; P2A] had no effect. The only system which showed significant differences in the effects of the catechols was the red blood cell. NDGA in the presence of H2O2 had a pronounced haemolytic effect, which did not correlate with its ability to induce methaemoglobin formation, while P2 had a much lower haemolytic effect, but stimulated the oxidation of haemoglobin to a greater extent than NDGA. NDGA is more hydrophobic than P2 which would result in a greater membrane effect. The pent-4-en-1-yne chain which links the catechol moieties in P2 can take part in the resonance structures of the semiquinone free radical, thus assisting in its stabilization and leading to increased methaemoglobin formation. This stabilization is also demonstrated by the fact that P2A affected the oxidation of haemoglobin to nearly the same extent as NDGA.

The mechanisms by which two anti-leprotic drugs (clofazimine and dapsone), both with anti-inflammatory properties, inhibit myeloperoxidase (MPO)-catalysed reactions, were investigated. The disappearance of NADH fluorescence was used as an assay for its oxidation. Chloride stimulated the oxidation of NADH in the MPO-H 2 O 2 system in a concentration-dependent manner (50-fold at 150 mM NaCl). Under these conditions Cl − is oxidized and the oxidant formed, presumably hypochlorous acid (HOC1), oxidizes NADH. Observations demonstrating the effect of the drugs on the MPO system, are; (1) Inhibition of Cl − -stimulated oxidation of NADH. (2) Inhibition of polypeptide modification in a model protein, thyroglobulin (TG). (3) Protection of MPO against loss of catalytic activity caused by chlorinating oxidants generated by the system. (4) Inhibition of haemoglobin oxidation. Only dapsone was active here. HPLC analyses suggested that the drugs were not significantly metabolized in the MPO-H 2 O 2 system in the absence of Cl − . Bleaching of clofazimine was stimulated by Cl − in the MPO system, suggesting the involvement of HOCl. Clofazimine was found to be a more potent scavenger of HOCl than dapsone when the inhibition of NADH oxidation by reagent HOCl was used as an assay. This finding is also supported by HPLC analyses which indicated a greater sensitivity of HOCl for clofazimine than for dapsone. Relatively low concentrations of dapsone inhibited the oxidation of oxygenated haemoglobin (HbO 2 ), suggesting that the drug was not metabolized to its N-hydroxylated derivative which is thought to be responsible for methaemoglobin (metHb) formation in vivo . It is proposed that the inhibitory mechanism of action of clofazimine is to scavenge chlorinating oxidants generated by the MPO-Cl − -H 2 O 2 system, while dapsone converts MPO into its inactive compound II (ferryl) form. The different inhibitory mechanisms of clofazimine and dapsone towards the MPO system may contribute to the anti-inflammatory actions of the drugs.

The univalent oxidation of chlorpromazine (CPZ) by the myeloperoxidase (MPO-H2O2 system led to the formation of a cation free radical (CPZ+) which was observed optically at 527 nm. CPZ protected MPO against loss of catalytic activity when co-oxidized in a MPO-Cl−-H2O2 system. Due to the stability of CPZ+ either further oxidation, or reduction back to the mother compound, become important mechanisms for disappearance of the free radical. Thus, the rate of formation and decay of CPZ+ were higher in the presence of Cl− than in its absence, since the radical can also be oxidized further by hypochlorous acid (HOC1), which is formed in the MPO-Cl−-H2O2 system. Decay of CPZ+ can also be due to electron acceptance from ascorbic acid or oxygenated haemoglobin (HbO2), resulting in regeneration of CPZ. When CPZ+ was generated in the MPO-H2O2 system, addition of HbO2 resulted in a sudden decrease in CPZ+ absorbance at 527 nm and a concomitant formation of metHb. When HbO2 was not added, the decay of CPZ+ was much slower. CPZ (in the absence of the MPO system) also stimulated the oxidation of HbO2 in the presence of 20 μM H2O2, but this reaction was considerably slower than when CPZ+ (generated by the MPO system) was allowed to react directly with HbO2. These results suggest that HbO2 was oxidized by CPZ+. To study the effect of CPZ intermediates, thyroglobulin (TG) was used as a model polypeptide. Chlorinated oxidants formed in the MPO system (in the absence of CPZ) induced TG peptide bond splitting. In contrast, CPZ metabolites generated by the MPO system (in the absence of Cl−) induced polymerization of TG, as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

The inhibition of arterial tone produced by two nitric oxide (NO) derivatives of biological relevance, dinitrosyl–iron complexes with cysteine (DNIC-CYS) or with glutathione (DNIC-GSH), was compared. Both compounds induced vasorelaxation within the same concentration range (3–300 nM) in endothelium-denuded rat aortic rings. Consistent with a faster rate of NO release from DNIC-CYS than from DNIC-GSH, the relaxant effect of DNIC-CYS was rapid in onset and tended to recover with time, whereas the one of DNIC-GSH developed slowly and was sustained. In addition, DNIC-GSH (0.3 and 1 μM) but not DNIC-CYS (1 μM) induced, even after washout of the drug, a persistent hyporesponsiveness to vasoconstrictors and a relaxant effect of low molecular weight thiols like N -acetylcysteine (NAC, which can displace NO from preformed NO stores). Both effects of DNIC-GSH were associated with elevation of cyclic GMP content and were attenuated by NO scavengers or a cyclic GMP-dependent protein kinases inhibitor. In rings previously exposed to DNIC-GSH, addition of mercuric chloride (which can cleave the cysteineNO bond of S -nitrosothiols) elicited relaxation, completely blunted the one of NAC and also abolished the persistent elevation of NO content. In conclusion, this study shows that whereas both DNIC-CYS and DNIC-GSH elicited a NO release-associated relaxant effect in isolated arteries, only DNIC-GSH induced an inhibition of contraction which persisted after drug removal. The persistent effect of DNIC-GSH was attributed to the formation of releasable NO stores in arterial tissue, most probably as S -nitrosothiols. Thus, the nature of the thiol ligand plays a critical role in determining the mechanisms and duration of the effect of LMW-DNIC in arteries.

The effect of amlodipine, a novel calcium channel blocker of the dihydropyridine type, on rabbit platelet aggregation, and the possible antiaggregatory mechanisms of amlodipine, especially on the nitric oxide (NO) guanosine 3',5'-cyclic monophosphate (cyclic GMP)-mediated pathway, were investigated. Other effects of amlodipine on thromboxane B2 (TXB2) formation in platelets also were examined. Amlodipine concentration-dependently inhibited rabbit platelet aggregation induced by collagen (10 microg/mL) or thrombin (0.1 U/mL) with an IC50 range of 32-69 microM. Along with this inhibition, our results also demonstrated that in the presence of L-arginine (100 IM), amlodipine (50 microM) increased nitric oxide synthetase (NOS) activity (from the resting activity of 2.05+/-0.36 to 7.11+/-0.95 pmol/mg protein/min) and NO release (by 80%), accompanied by an elevation of the cyclic GMP level (from the resting platelet level of 1.27+/-0.12 to 6.21+/-0.55 pmol/10(9) platelets) induced by collagen (10 microg/mL). However, the antiaggregatory effect of amlodipine (50 microM) could be attenuated significantly by oxyhemoglobin (5 microM), a NO scavenger, or N(G)-nitro-L-arginine methyl ester (100 microM), a specific NOS inhibitor. In addition, the TXB2 production in platelets induced by collagen or thrombin was concentration-dependently inhibited by amlodipine. Therefore, we propose that the antiaggregatory mechanisms of amlodipine might be mediated, in part, by a NO-cyclic GMP process accompanied by the inhibition of TXB2 formation in platelets.

To estimate the reaction of two primary redox-related species of nitric oxide (i.e. NO+ vs NO*) from a variety of NO donors, we employed the differential interactions of these NO forms with oxyhemoglobin (oxyHb) as a chemical assay. NO+ formation was estimated by the S-nitrosation reaction with oxyHb, and NO* formation via its reaction with the oxygen-heme complex of oxyHb. Under the conditions employed, all NO donors caused concentration-dependent formation of methemoglobin, indicative of NO* liberation. However, the extent of S-nitrosation was substantially different among the NO donors studied. A representative S-nitrosothiol, S-nitroso-N-acetyl-penicillamine, caused significantly more S-nitrosation than nitroglycerin, isobutyl nitrite, sodium nitroprusside, and 3-morpholino-sydnonimine (ANOVA, P < 0.05). These results indicated that NO donors can differ in their interactions with oxyHb, and possibly with other target proteins, in part because they liberate or transfer different ratios of NO redox forms. This difference may contribute, in part, to the diversity of pharmacological effects elicited by NO donors.

As previously shown with isolated oxyhemoglobin, methemoglobin formation can also be induced in intact erythrocytes by hydroxylamine compounds and substituted phenols such as butylated hydroxyanisole (BHA). Electron spin resonance investigations revealed that, accordingly, free radical intermediates were formed in erythrocytes from hydroxylamine, N,N-dimethylhydroxylamine, and N-hydroxyurea. Due to the low stability of the dihydronitroxyl radicals, their detection required the use of a continuous flow system and relatively high amounts of the reactants. As has already been demonstrated with the solubilized hemoglobin system, hemoglobin of intact erythrocytes also reacts with the more hydrophilic xenobiotics such as hydroxylamine. However, the reaction rate was slightly reduced, indicating the existence of an incomplete permeability barrier for these compounds. The limited solubility of phenolic compounds in the aqueous buffer of suspended erythrocytes (in combination with the strict requirement of osmolarity in order to prevent hemolysis) impeded the direct detection of the respective phenoxyl radicals previously reported in hemoglobin solutions. However, in accordance with earlier findings in homogeneous reaction systems, chemiluminescence was observed as well, indicating the existence of a further reaction intermediate, which was also obtained in pure hemoglobin solutions when mixed with the respective reactants. As has recently been demonstrated, this light emission is indicative of the existence of highly prooxidative compound I intermediates during methemoglobin formation. Prooxidant formation in erythrocytes is reflected by a significant decrease in thiol levels even with those compounds where free radical formation was not directly detectable by ESR spectroscopy. The use of the spin-labeling technique revealed membrane effects as a result of oxidative stress. Oxidative metabolism of hemoglobin with hydroxylamine caused a release of low molecular weight iron. The marked hemolysis observed in the presence of BHA results from a direct membrane effect of this compound rather than a consequence of free radical-induced oxidative stress. A correlation of the different results is discussed in terms of possible toxicological consequences.

The metabolic activation of halothane by human haemoglobin (Hb) under reducing conditions in vitro is reported. Absolute spectra of sodium dithionite-reduced Hb, recorded during its anaerobic incubation in the presence of the substrate, showed decreasing concentrations of reduced Hb (Hb 2+ ) with time. The loss of Hb 2+ was accompanied, although only to some extent, by a concurrent oxidation to methaemoglobin (Hb 3+ ), suggesting that electron transfer from Hb to the substrate had occurred. Reductive halothane metabolism was observed under these conditions as indicated by a dose-dependent inorganic fluoride (F − ) production, which was, however, lower than that observed with heated Hb or a water soluble haem preparation (methaemalbumin). A rapid, partial loss of Hb was found upon addition of the substrate to the incubation mixture, as indicated by a decrease of the typical peak at 418 nm in the absolute spectra recorded in the presence of carbon monoxide (CO). This effect was associated with a loss of the Hb prosthetic group, haem, as shown by a decrease of the pyridine-haemochromogen reaction. Both effects were time and dose dependent. The inhibition of the Hb inactivation reaction by adding exogenous CO or the spin trapping agent N - t -butyl- α -phenylnitrone (PBN) to the incubation mixture beforehand indicated that (a) a reduced and free haem iron is required by Hb to activate halothane, and (b) the formation of free radical reactive metabolites of halothane is likely to be responsible for Hb inactivation. The mechanism of the reaction may involve the attack of these metabolites on the haem group of Hb, as indicated by the detection, with a reverse-phase ion-pairing HPLC system, of two Hb-derived products showing a typical haem-like absorption spectrum. The present results resemble those obtained recently with carbon tetrachloride (Ferrara etal., Alternatives to Laboratory Animals 21 : 57–64, 1993) and suggest a common mechanism of activation of the two polyhalogenated alkanes by Hb.

We have studied the efflux of dapsone hydroxylamine from normal and diabetic erythrocytes by the use of a two-compartment (1 and 2) in vitro dialysis system, in order to model the in vivo blood supply to the bone marrow. When both types of erythrocytes were dialysed against mononuclear leucocytes, the hydroxylamine crossed the membrane and caused significantly greater white cell death compared with dialysis of leucocytes against untreated erythrocytes. However, in the case of both normal and diabetic cells, the presence of the glutathione depletor diethyl maleate (DEM) caused a marked reduction in movement of hydroxylamine from compartment 1 to 2. Diethyl dithiocarbamate (DDC), a methaemoglobin accelerant, caused a marked reduction in movement of hydroxylamine from erythrocytes (diabetic and normal) in compartment 1 to 2 which led to a significant reduction in white cell death compared with the absence of DDC (18.3 +/- 5.5 vs 34.8 +/- 8.1%, P0.05). Dapsone recovery from compartment 1 rose significantly in the presence of DDC compared with control in both erythrocyte types. In contrast, recovery of dapsone from normal erythrocytes incubated in compartment 1 was significantly reduced by the presence of DEM compared with control, although there was no difference between control and DEM-treated diabetic cells. Dapsone analysis in compartment 2 revealed a significant increase in dapsone recovery in both diabetic (11.3 +/- 1.1%) and normal (11.9 +/- 1.1%) erythrocytes in the presence of DDC compared with diabetic (3.3 +/- 0.4%) and normal control (4.8 +/- 2.0%, P0.001). The presence of DEM in compartment 1 caused a significant fall in dapsone recovery in compartment 2 (3.7 +/- 0.26) compared with control (4.7 +/- 0.36%, P0.05). Hence, dapsone hydroxylamine is capable of leeching out of normal and diabetic erythrocytes, traversing a semipermeable membrane and causing toxicity to human mononucleocyte cells in vitro. This process may be one of the first stages in immune-mediated agranulocytosis.

The fate of dapsone hydroxylamine has been investigated in diabetic and normal human erythrocytes. In erythrocytes from four type 1 (insulin dependent) diabetic subjects, there was a significant decrease in dapsone hydroxylamine-mediated methaemoglobin formation compared with cells drawn from normal individuals (P < 0.01). However, the ability of the diabetic cells to detoxify the hydroxylamine to dapsone was not correspondingly reduced and was not different to normal cells. The initial rate of the accelerating effect of diethyl dithiocarbamate (DDC) on hydroxylamine-mediated methaemoglobin and dapsone formation was significantly reduced in diabetic compared with normal cells. There was no significant difference in hydroxylamine-dependent methaemoglobin formation between diabetic erythrocytes pretreated with either statil or sorbinil and untreated diabetic cells. Dapsone recovery in diabetic erythrocytes incubated with statil was not significantly different from statil-free incubations. However, in the presence of sorbinil, there was a marked reduction in dapsone formation at all four time points, (P < 0.001 at 15 min). Mean measured levels of glutathione did not differ significantly between the normal (380 ± 30.9 mg/L; N = 8) and diabetic (349 ± 58.7 mg/L; N = 8) volunteers. In summary, although diabetic erythrocytes were less sensitive to the effect of dapsone hydroxylamine-mediated methaemoglobin formation in comparison with normal cells, glutathione-dependent hydroxylamine reduction to dapsone was unaffected.

The oxidative activities of primaquine [6-methoxy-8-(4-amino-1-methylbutylamino)quinoline] and its metabolites, the quinone-imine derivatives of 5-hydroxyprimaquine [5-hydroxy-6-methoxy-8-(4-amino-1-methylbutylamino)quinoline] and 5-hydroxydemethylprimaquine [5-hydroxy-6-demethyl-8-(4-amino-1-methylbutylamino)quinoline], 6-methoxy-8-amino quinoline and hydrogen peroxide, were studied on rat erythrocytes in vitro and in vivo. In both cases, the most effective metabolites in oxidizing hemoglobin and depleting non-protein sulfhydryl groups from erythrocytes were the quinone-imine derivatives of the ring-hydroxylated metabolites, 5-hydroxyprimaquine and 5-hydroxydemethyl-primaquine. The latter quinone-imines were shown by light absorption spectroscopy and oxygen consumption studies to be able to oxidize purified rat hemoglobin to methemoglobin but to be unable to react directly with reduced glutathione. In agreement with these results, no radical adduct was detected by electron paramagnetic resonance spectroscopy in incubations of rat erythrocytes with the quinone-imines and the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide; metabolite-derived free radicals were detected instead. Taken together, the results suggest that 5-hydroxyprimaquine and 5-hydroxydemethylprimaquine are important metabolites in the expression of primaquine hemotoxicity, in contrast to 6-methoxy-8-aminoquinoline. Additionally, the results indicate that hydrogen peroxide is the ultimate oxidant formed from the ring-hydroxylated metabolites by redox-cycling of the corresponding quinone-imine derivatives both in vitro and in vivo.

We have used an in vitro two-compartment model, to investigate the ability of dapsone, formed by erythrocyte-mediated detoxification of its hydroxylamine metabolite, to escape the cells and cross a semi-permeable membrane into both plasma and other erythrocytes. Both diethyl dithiocarbamate (DDC) treated and untreated erythrocytes were incubated with dapsone hydroxylamine and dialysed against either fresh cells or plasma. Methaemoglobin was predominantly detectable in compartment A although the presence of low levels of methaemoglobin in compartment B indicated that the hydroxylamine itself had crossed the membrane. In contrast to methaemoglobin disposition, recovery of dapsone was higher (P0.05) in compartment B compared with A for all three treatment groups at 30 and 60 min, but not at the remaining time points. Regression analysis of the cumulative recovery of dapsone over 150 min in all three treatment groups for both compartments A and B showed correlation coefficients close to unity. In compartment A, analysis of the mean slopes of the regression lines indicated that, overall, significantly more dapsone was recovered from group 1 (erythrocytes, hydroxylamine and DDC dialysed against untreated red cells) compared with group 3 (erythrocytes and hydroxylamine dialysed against plasma) (0.22 +/- 0.05 vs 0.09 +/- 0.005; P0.025). Also in compartment A, significantly more dapsone was recovered from group 2 (erythrocytes and hydroxylamine dialysed against untreated red cells) compared with group 3 (erythrocytes and hydroxylamine dialysed against plasma: 0.16 +/- 0.02 vs 0.09 +/- 0.005). In compartment B, dapsone recovery was significantly greater in group 1 (erythrocytes, hydroxylamine and DDC dialysed against untreated red cells; slope of regression line: 0.59 +/- 0.05) compared with group 2 (erythrocytes and hydroxylamine dialysed against untreated red cells; slope of line: 0.28 +/- 0.02, P0.005). In addition, dapsone recovery was significantly greater in group 1 (0.59 +/- 0.05) compared with group 3 (erythrocytes and hydroxylamine dialysed against plasma; 0.21 +/- 0.02, P0.005). Dialysis of erythrocytes with dapsone itself over 120 min caused no detectable methaemoglobin formation. The process of erythrocyte-mediated dapsone formation from its hydroxylamine may feasibly occur in vivo and contribute to the systemic persistence and therapeutic effect of dapsone.

Red blood cell cytosol promotes enzymic N-demethylation reactions which display typical Michaelis-Menten kinetics with respect to N-methylaniline as substrate. The demethylase activity is linked with hemoglobin (Hb) and is enhanced in the presence of NADH and the NADH-methemoglobin reductase system. It has been adduced that Hb in its oxygenated form is involved in the reaction.

Mechanistic aspects of the reaction of hydrogen peroxide with methemoglobin with respect to phenothiazine oxidation have been studied. Three phenothiazines, methoxy- (MoPZ), chlor- (CPZ) and methoxycarbonylpromazine (MaPZ), have been used. These phenothiazines differ only in substitution at the 2-position, which contributes substantially to the electron-donating properties of these compounds. Reaction with hydrogen peroxide oxidizes methemoglobin to ferrylhemoglobin, which contains iron(IV)-oxo porphyrin moiety and a protein radical. The phenothiazines are oxidized by ferrylhemoglobin in the presence of H2O2 mainly to their sulfoxides, with a radical cation as intermediate. The conversion rates (MoPZ greater than CPZ greater than MaPZ) decrease with the electron-withdrawing ability of the 2-substituent, as indicated by Hammett sigma para values. Hydrogen peroxide consumption during the reaction is similar for the three phenothiazines. Denaturation reactions that occur upon exposure of methemoglobin to hydrogen peroxide have been investigated. For this heme-protein cross-linking was studied by means of heme retention by the protein after methyl ethyl ketone extraction. Furthermore, oxygen consumption during the reaction was assayed, which indicates formation of protein-peroxy radicals. The extent of both heme-protein cross-linking and oxygen consumption is decreased by phenothiazines in the same order as the phenothiazine conversion rate. CPZ sulfoxide is not converted by methemoglobin in the presence of hydrogen peroxide, and CPZ sulfoxide shows no effect on heme-protein cross-linking and oxygen consumption. The results are explained by electron transfer from phenothiazine to the protein radical. Stronger electron donors (MoPZ greater than CPZ greater than MaPZ) are converted faster and by reducing the protein radical they better protect hemoglobin against denaturation. A catalytic cycle, that takes into account our observation and the existing knowledge of hemoglobin oxidation states, is presented.

Micromolar concentrations of diethyldithiocarbamic acid (DDC) kill fungi, bacteria and malaria. DDC forms chelates with copper and the microbicidal effectiveness of this drug is enhanced greatly by small amounts of copper. DDC, in the presence of at least 1 molar equivalent of copper, also causes lysis of human erythrocytes. To explore the cytocidal actions of DDC and copper, we have used human erythrocytes and Escherichia coli as models. We found that: (1) the combination of DDC and copper also lysed E. coli spheroplasts, suggesting a possible common mechanism of hemolytic and microbicidal action; (2) higher ratios of drug: metal (greater than 4:1) diminished hemolytic and, as observed earlier, microbicidal effects; (3) cobalt, known to suppress the microbicidal effects of DDC:Cu, also prevented red cell lysis; (4) despite the necessary involvement of copper in DDC-mediated hemolysis, there was no evidence of oxidative damage to erythrocytes, and both lysis of erythrocytes and killing of E. coli were undiminished in the absence of oxygen; (5) the DDC:Cu chelate preferentially located in organic solvents and in membranes of erythrocytes. The chelate was quite soluble in chloroform but much less so in a C-16 hydrocarbon (hexadecane) which resembled erythrocyte membrane lipid. In hexadecane and at greater than 10(-4) M DDC and 5 x 10(-5) copper, an amphipathic drug:metal complex accumulated at the organic:aqueous interface; and (6) this amphipathic complex may permeabilize the lipid bilayer, causing leakage of ions and cell water and eventuating in colloid osmotic lysis. Red cells and E. coli exposed to the chelate showed early loss of intracellular rubidium (86Rb+). Furthermore, lysis of erythrocytes and E. coli spheroplasts was suppressed by the inclusion of either dextran or sucrose. Thus, it appears that DDC:Cu chelates are cytocidal by virtue of concentrating in the lipid bilayer and, perhaps, forming amphipathic complexes which disrupt membrane integrity. Drugs with similar behavior hold promise for therapy of malaria because metals capable of forming such complexes may accumulate within parasitized red cells.

N. Hydroxyurea forms methemoglobin from oxyhemoglobin with concomitant formation of the aminocarbonylaminooxyl radical H2N-CO-NHO., as detected with electron paramagnetic resonance spectroscopy (EPR). This radical could be detected for several hours in a low steady-state concentration. Approximately 1 hr after the reaction had been started, the EPR spectra of two additional paramagnetic intermediates could be detected at low temperature (77 degrees K), a low-spin ferric methemoglobin complex with hydroxyurea (MetHb-NHOH-CO-NH2) and the hemoglobin-nitric oxide adduct (Hb2(+)-NO). The intensities of their EPR spectra increased steadily over the range of more than 64 hr. The low-spin ferric methemoglobin complex was immediately formed when hydroxyurea was dissolved in a methemoglobin whereas the nitric oxide complex was possibly an oxidation product of the MetHb-hydroxyurea adduct. Its oxidative degradation is known to lead to the very toxic compounds nitric oxide and nitrogen dioxide which can therefore contribute to the toxic action of hydroxyurea.

The effects of the primaquine (PQ) enantiomers, (+)PQ and (-)PQ, and two putative metabolites [5-hydroxyprimaquine (5HPQ) and 6-desmethyl-5-hydroxyprimaquine (6D5HPQ)] on methemoglobin (Met Hb) and glutathione content and release of hemoglobin into plasma from glucose-6-phosphate dehydrogenase (G-6-PD) deficient red cells were studied in vitro. The results show that a 1.5 mM concentration of (-)PQ produced a significantly greater increase in Met Hb content and decrease in reduced glutathione (GSH) level than did (+)PQ. However, the release of plasma hemoglobin was greater with (+)PQ than with (-)PQ. The hydroxy derivatives of primaquine, 5HPQ and 6D5HPQ, were significantly more active than PQ. Their individual effects differed; whereas 5HPQ produced significantly greater reduction in GSH compared to 6D5HPQ, the effect of 6D5HPQ on Met Hb content and release of plasma hemoglobin was greater than that of 5HPQ. The qualitative effects of these compounds on normal, heterozygous and hemizygous G-6-PD deficient red cells were similar, but quantitatively the effects were greatest on hemizygous G-6-PD deficient cells and intermediate on heterozygous cells.

Oxidation of the antimalarial primaquine by horseradish peroxidase and H2O2 was demonstrated by visible light absorption and ESR spectroscopy. Initial product analysis indicated a 15% yield of O-demethoxylation products, methanol and the quinone-imine derivative, and organic extractable polymeric material. Horseradish peroxidase was substituted by methemoglobin, and both enzymes showed greater activity at acidic pH values. During the enzymatic oxidation of primaquine, a drug-derived free radical was detected by direct ESR spectroscopy. A similar ESR spectrum was obtained during enzymatic oxidation of 6-hydroxyprimaquine at pH 9.0. Computer simulations of the ESR spectra obtained in normal and deuterated buffer indicated that the detectable free radical contains two primaquine moieties. This in vitro oxidation of primaquine to a free radical intermediate that is stable in the presence of oxygen might be considered a new mechanistic route for analyzing the pharmacological effects of primaquine.

Suspensions of washed human red blood cells were treated with nine synthetic putative metabolic derivatives of primaquine (PQ'), and their individual effects on activity of the hexose monophosphate shunt (HMS) were quantitated by radiometric analysis of 14CO2 from [14C] glucose. The most potent HMS stimulant was 5-hydroxy-6-methoxy-8-aminoquinoline (5H6MQ), which caused 10-fold elevation of HMS activity at an estimated concentration of 0.004 mM. Ten millimolar primaquine (PQ) was required to achieve the same effect. Thus, 5H6MQ was approximately 2500-fold more reactive with the HMS than PQ. Other analogs achieved less than 0.4- to 154-fold increases in HMS reactivity. Patterns of effects on HMS activity indicated that 5-hydroxylation and/or N-dealkylation of PQ strongly enhanced HMS reactivity. In contrast, none of the putative metabolites of PQ activated the proteolytic system known to degrade oxidized protein in red cells, indicating that stimulation of the HMS by the PQ analogs was not related to an injurious oxidative stress. Red cells pretreated with 1.0 mM N-ethylmaleimide (NEM) or with 1.0% (w/v) sodium nitrite to cause glutathione sulfhydryl blockage and conversion of red cell hemoglobin to methemoglobin (metHb), respectively, also showed elevation of HMS activity when exposed to 5H6MQ. These observations suggested that 5H6MQ-induced elevation of HMS activity was at least partially independent of glutathione redox reactions, hydrogen peroxide accumulation and reaction with oxyhemoglobin. The relevance of these observations to proposed mechanisms of hemolytic toxicity of PQ is discussed.

The possible role of hemoglobin in the sulfoxidation of chlorpromazine is still a controversial subject. Therefore this sulfoxidation was investigated with purified oxyhemoglobin and methemoglobin under various conditions: (i) in phosphate buffer pH 6.5; (ii) in monooxygenase mimicking systems with electron donors like ascorbic acid and NADPH, the last, with and without an electron carrier like methylene blue and cytochrome c reductase; (iii) in the presence of H2O2. Only in the presence of H2O2 chlorpromazine was converted into chlorpromazine sulfoxide in a considerable amount. This so-called peroxidase activity of hemoglobin appeared not to be based on a Fenton-type reaction. An oxidized reactive form of hemoglobin (i.e. ferrylhemoglobin) is responsible for the sulfoxidation. In the other systems only with ascorbic acid some chlorpromazine sulfoxide was produced. This is probably due to the production of H2O2 and the subsequent peroxidase activity of hemoglobin. Chlorpromazine enhanced the autoxidation of oxyhemoglobin, without being transformed itself.

Human placental microsomes prepared by conventional methods were compared with analogous preparations from adult, male rat livers with respect to biochemical components and systems which could affect rates of mixed-function oxidation and reduction of drugs and steroids in vitro . Each of the electron transport components required for the mixed-function oxidation of drugs in hepatic systems was present in lower concentrations in placental than hepatic microsomes. In contrast to hepatic microsomes, placental microsomes which exhibited unusually high aryl hydrocarbon hydroxylase activities did not contain increased concentrations of the electron transport components. Evidence was provided to indicate that rapid degradation of initial electron donors (reduced pyridine nucleotides) was not responsible for the observance of low or negligible drug metabolic activities observed in incubations with placental micro-somes. Cytochromes P-450 and b 5 and their corresponding reductases were shown to be present in human placental preparations. NADPH and NADH-dependent cytochrome c reductase activities in placental microsomes were somewhat lower but comparable to those determined in hepatic preparations. However, cytochromes b 5 and P-450 and contaminating hemoglobin and methemoglobin accounted for less than 56 per cent of the total heme present in placental microsomes. Rapid degradation of placental cytochrome P-450 was observed at 37° in the presence of sodium hydrosulfite, but conversion to cytochrome P-420 was minimal after incubation for 1 hr in the presence of NADPH at the same temperature. It was con- sidered probable that the low rates of drug biotransformation observed would be explicable in terms of high substrate specificities of the placental enzymic components.

Human red blood cells treated with the CuZn superoxide dismutase inhibitor diethyldithio-carbamate (DDC) undergo metabolic modifications in addition to the superoxide dismutase inhibition: oxidation of the reduced glutathione (GSH) to oxidized glutathione (GSSG), methemoglobin formation, and increased hexose monophosphate shunt activity were observed. The magnitudes of these changes are dependent on the DDC concentration. Under nitrogen, only superoxide dismutase inhibition occurs. After removal of the GSH with N -ethylmaleimide, production of H 2 O 2 can be detected by measuring the red cell catalase inhibition in the presence of 3-amino-1,2,4-triazole. H 2 O 2 production is not altered by conversion of oxyhemoglobin to methemoglobin by sodium nitrite prior to incubation. GSH oxidation and methemoglobin formation are stopped when DDC is eliminated from the incubation medium after completion of the superoxide dismutase inhibition. These data indicate that methemoglobin formation and modification of the GSH status in red cells treated by DDC are not a direct consequence of the CuZn Superoxide dismutase inhibition but are due rather to a DDC-dependent production of H 2 O 2 .

Red cells suspended in either defined medium or buffered plasma were oxidatively stressed by incubation in the presence of 1, 4-naphthoquinone-2-sulfonate at concentrations which caused less than 50% methemoglobin accumulation, stimulation of the hexose monophosphate shunt to less than 15% of capacity, and about a 30% increase in flux through glycolysis. Normal plasma concentrations of lactate and pyruvate in either defined medium or buffered plasma allowed increased contribution of reducing equivalents from glycolysis in response to oxidative stress. Increased utilization of reducing equivalents by the red cell was observed as increased accumulation of pyruvate, whereas accumulation of lactate represented storage of reducing equivalents. Exogenous lactate or pyruvate did not serve as a net electron source or sink since the total content in red cell suspensions of both lactate and pyruvate was increased during exposure to oxidative stress. If exogenous lactate had been used as a net source of reducing equivalents, the lactate concentration would have decreased during incubation of red cell suspensions. Plasma ascorbate or other constituents did not alter the qualitative response of glycolysis to oxidative stress (decreased lactate accumulation, increased pyruvate accumulation, and increased total flux through glycolysis), but plasma constituents did raise significantly the dose of oxidant agent required to elicit a given quantitative response. At levels of oxidative stress likely to be encountered in vivo, glycolysis and the hexose monophosphate shunt may be equal in importance as aerobic/antioxidant pathways.

Stimulation of the hexose monophosphate shunt by primaquine results from the oxidation of NADPH by primaquine. This conclusion was based on the observations that primaquine lowered cellular NADPH but not GSH and that, in red cells in which the GSH was unavailable for reaction, primaquine still stimulated the rate of the hexose monophosphate shunt. In a non-cellular system, primaquine interacted with NADPH, but not GSH, to produce H2O2. Stimulation of the hexose monophosphate shunt by primaquine does not primarily involve H2O2 accumulation since stimulation of the pathway by primaquine was also observed in red cells containing methemoglobin, a red cell preparation in which no H2O2 accumulates. Methemoglobin prevented the formation and/or accumulation of H2O2 in intact red cells incubated with primaquine as well as in a non-cellular system containing primaquine plus Fe2+-EDTA as an H2O2 source. Methemoglobin probably acts by scavenging reactive intermediates since oxyhemoglobin was formed from methemoglobin in the non-cellular experiments. In the red cell, primaquine stimulated glucose-dependent conversion of methemoglobin to oxyhemoglobin.

Methemoglobin formation by nitroso and amino derivatives of chlorinated biphenyl ether was investigated. All the nitroso derivatives used induced methemoglubin formation in suspensions of human erythrocytes. Ability to induce methemoglobin formation among the nitroso derivatives was the highest for nitrosobenzene, followed by p -nitrosobiphenyl ether, 4-chloro p -nitrosobiphenyl ether, 2,4-dichloro p -nitrosobiphenyl ether and then 2,4,6-trichloro p -nitrosobiphenyl ether. The monochloro p -nitrosobiphenyl ethers showed all the same activity. Therefore, the ability to induced methemoglobin formation depended on the number of chlorines rather than on the position of chlorine substitution on the phenoxy group in the molecule. Since lactate decreased but glucose and xylitol increased the methemoglobin level produced by the nitroso compounds, this reaction would appear to require an NADPH-dependent enzyme system in erythrocytes. All of the amino derivatives used induced methemoglobin formation in suspensions of human erythrocytes with rat liver homogenates. Ability to induced methemoglobin formation among the amino derivatives was the highest for p -aminobiphenyl ether, followed by 4-chloro p -aminobiphenyl ether, 2,4,-dichloro p -aminobiphenyl ether, 2,4,6-trichloro p -aminobiphenyl ether, and then aniline. Methemoglobin formation bychlorinated p -aminobiphenyl ether was examined by intraperitoneal and oral administration to rats. After i.p. injection, methemoglobinemia was induced by p -aminobiphenyl ether, aniline, and 2,4-dichloro p -aminobiphenyl ether but not by 2,4,6-trichloro p -aminobiphenyl ether over the period tested. The order of methemoglobin formation was p- aminobiphenyl ether ≧ 4- chloro p- aminobiphenyl ether > aniline ≧ 2,4- dichloro p -aminobiphenyl ether. Oral administration gave almost the same result as i.p. injection. The maximum methemoglobin levels produced by the amino derivatives were highest for 4-chloro p -aminobiphenyl ether, followed by p -aminobiphenyl ether, 2,4-dichloro p -aminobiphenyl ether, aniline, and then 2,4,6-trichloro p -aminobiphenyl ether. However, the methemoglobin level produced by 2,4,6-trichloro p -aminobiphenyl ether was very low and not significantly greater than the background level.

4,4′-Diaminodiphenylsulfone (DDS) can produce methemoglobinemia in vivo , apparently by the action of an unidentified metabolic product. To discern the chemical nature of such a compound, 13 p -substituted diphenylsulfones have been examined for their ability to oxidize hemoglobin (Hb) to methemoglobin (MHb) in human erythrocytes in vitro . 4-Amino-4′-hydroxyaminodiphenylsulfone (DDS-NOH) is most effective, 1·9 × 10 −5 M of drug oxidizing as much as 60 per cent of the Hb, or 100 heme equivalents/mole of DDS-NOH in 2 hr. Oxygen is required for this process, and glucose, while not required, enhances MHb formation. With dialyzed hemolysates and solutions of purified Hb, the heme equivalents oxidized/mole of drug becomes less than 10, but a ratio of 80 : 100 can be restored by adding reduced glutathione (GSH) or NADPH. Apparently DDS-NOH produces MHb by coupled oxidation with Hb and O 2 and DDS-NOH can be regenerated by reducing compounds to yield the high heme oxidation ratio. In intact cells, DDS-NOH causes a 7-fold increase in the activity of the hexose monophosphate shunt as measured by 14 CO 2 production from glucose 1- 14 C. Incubation with galactose, which is minimally oxidized by the shunt pathway, results in decreased formation of MHb, and the GSH concentration drops by 75 per cent. Depletion of intracellular GSH by a sulfhydryl oxidizing compound also reduces MHb formation by DDS-NOH. These observations implicate GSH or NADPH or both in the recycling of DDS-NOH in erythrocytes. Consistent with these interpretations, DDS-NOH produced only one-half as much MHb in erythrocytes from three individuals with glucose 6-phosphate dehydrogenase deficiency as it did in normal erythrocytes.

Sodium nitrite induced equivalent levels of methemoglobin in washed erythrocytes from cat, dog, and man, all suspended in Krebs-Ringer phosphate-glucose (pH 7·4). The same levels occurred in human cells with or without aded substrate (glucose or lactate). In all these incubations, reduction of methemoglobin was minimal or absent over a 2-hr period. When 10−5M methylene blue was added with glucose, equivalent increases in rates of methemoglobin reduction occurred in the cells of all three species. Similar rates were seen in rabbit and mouse red cells even without added methylene blue, as long as lactate or glucose was present. Methylene blue further enhanced reductase activity in mouse cells but only in the presence of glucose. Rabbit cells responded much less dramatically, if at all, to methylene blue. Lysates of human, rabbit, and mouse cells were equally sensitive to nitrite, and no spontaneous reduction occurred. These findings suggest that the high reductase activity of rabbit and mouse erythrocytes is NADH-dependent. The mouse but not the rabbit appears to possess also a NADPH-dependent reductase like man, dog and cat.

The formation of methemoglobin caused by phenacetin and acetanilide in rats is inhibited by simultaneous treatment with if Bis-[p-nitrophenyl] phosphate (BNPP), a new carboxylesterase inhibitor, while the methemoglobinemia after p-phenetidine is not influenced by BNPP. The duration of BNPP action on phenacetin-induced methemoglobinemia and the spontaneous return of liver esterase activity after a single dose of BNPP have been studied. The enzymatic hydrolysis of the amide bond of phenacetin by rat liver, in vivo and in vitro, is inhibited considerably less by BNPP than the hydrolysis of acetanilide, monoethylglycine-2,6-xylidide and methyl butyrate, and another amidase may be involved in the deacetylation of phenacetin by rat liver.

It has previously been demonstrated, in experiments using the isolated perfused rat liver, that amylase is synthesized by the liver and then secreted into the plasma of the perfusing blood. Under normal conditions, production of amylase is about 30–40 units per g of liver per 4-hr perfusion period. Addition of 1–2 mg potassium cyanide (2–4 × 10 −4 M final concentration) to the perfusing blood had relatively little effect on amylase production. Addition of 5 mg KCN (10 −8 M) reduced amylase production to 11.5units/g of liver and addition of 10 mg resulted in a net loss of amylase in the 4-hr perfusion. Since 2 mg KCN is enough to kill a rat of the size from which these livers were taken, the amounts of cyanide necessary to inhibit amylase production are surprisingly high. It is postulated that the high rhodanese concentration in liver serves as a protective mechanism against the cyanide toxirity. Production of methemoglobin or the presence of thiosulfate in the perfusing blood protected against the inhibiting action of cyanide on amylase production.

In the rat acetophenetidin causes methemoglobinemia, which reaches a plateau in 2 hr. The formation of methemoglobin by acetophenetidin is diminished by barbiturates. In equieffective doses of several barbiturates—phenobarbital, hexobarbital, pernocton® = 5-(2-bromallyl)-5-(1-methyl-propyl)-barbituric acid and eunarcon® = 5-(2-bromallyl)-1-methyl-5-isopropylbarbituric acid—the maximal production of methemoglobin was decreased by 2 3 . Inhibition of the metabolic transformation of acetophenetidin may be responsible and consequently the treatment with barbiturates cause an increase in the serum concentration of acetophenetidin. Since the methemoglobin formation induced by p-phenetidin was also inhibited by barbiturates, the decline in methemoglobin caused by barbiturates may be due to an inhibition of the N-hydroxylation.

Evidence consistent with the formation of a reversible complex between excess free nitrite ion and the ferric heme groups of methemoglobin is presented. The existence of this complex in nitrited red cells or in lysates in the presence of appropriate concentrations of free nitrite can lead to gross underestimation of methemoglobin levels by a common spectrophotometric method. Complex formation is manifested by a decrease in the absorption of methemoglobin solutions in the range of 600–650 mμ. This source of error can be eliminated in red cell suspensions simply by washing. Similarly, lysate solutions may be dialyzed or diluted to effect dissociation of the complex. Attempts were made to estimate the dissociation constant of this complex at 25°, pH 7.4, and physiologic ionic strength. Although unknown side reactions between methemoglobin and nitrite interfere in the spectrophotometric determination of this constant, its value is probably not greater than 3 mM. This estimate indicates that the complex must exist transiently in vivo when mice receive nitrite doses adequate to convert more than one-third of the circulating blood pigment to methemoglobin. Thus, complex formation may contribute to the unusually sustained methemoglobinemia produced in mice by nitrite.

Microsomal cytochrome P450, the ethyl isocyanide difference spectrum of dithionite-reduced microsomes (measured at the pH giving equal absorbance of 430 and 455 mμ peaks), and the aniline type II difference spectrum were affected similarly after phenobarbital induction and during the subsequent aging in vitro (about 90 hr at 1° under nitrogen) of hepatic microsomes from rats pretreated with phenobarbital. In these same microsomal preparations from phenobarbital-pretreated rats, type I spectra (caused by adding hexobarbital or (+) benzphetamine to microsomes) were increased to a greater extent and were less stable to aging in vitro than the previously mentioned components. During aging, decreases in the type II spectra paralleled the loss of total heme, but large decreases in the type I spectra were observed with little or no loss of heme. Also, aging did not change the pH giving equal absorbance of the 430 and 455 mμ peaks in the ethyl isocyanide difference spectrum, nor was there any shift in the wavelength of the maximum (450 mμ) in the carbon monoxide difference spectrum of dithionite-reduced microsomes. The binding associated with type I or II spectral changes did not appear to conform to simple Michaelis-Menten kinetics. Plots of ΔA vs. ΔA/S (where ΔA is the difference in absorbance between the trough and the peak in the 350 to 450 mμ region of the type I or II substrate-induced microsomal difference spectrum, and S is the corresponding substrate concentration) exhibited curvature (concave upwards). During aging, the curvature associated with plots of ΔA vs. ΔA/S using ( + ) benzphetamine increased, while the curvature associated with plots of ΔA vs. ΔA/S using aniline decreased. Some correlation was observed between increases in the rate of N -demethylation of (+) benzphetamine or p -hydroxylation of aniline and the increases in the respective type I or II difference spectra, after phenobarbital treatment of rats. Also it appeared that the rate of p -hydroxylation of aniline decreased to a lesser extent on aging than the rate of N -demethylation of (+) benzphetamine.

Phenylhydrazine hydrochloride, p -Aminopropiophenone and Methylene blue serve as models for the examination of the temporal connexions between the possible formation of methemoglobin and the appearance of Heinz bodies. It is proved that there is no correlation between the formation of ferrihemoglobin and precipitations in the erythrocytes. These findings and the application of the various substances and the appearance of the Heinz bodies lead to the conclusion, that different mechanisms of the metabolism are responsible for the appearance of the Heinz bodies. The appearance of ferrihemoglobin is not a prerequisite for the presence of precipitations in the erythrocytes but can be observed in certain cases contemporary with or prior to the formation of Heinz bodies.

The effects of glucose oxidase, which oxidizes glucose and produces H2O2, were studied in mice after intraperitoneal injection of supralethal doses of the enzyme. Such injections cause the appearance of hematin pigment in the plasma, up to 20 per cent of this pigment being methemoglobin. After high lethal doses of glucose oxidase, the hematocrit decreases (readings as low as 7 have been obtained), and the total hematin pigment of whole blood also decreases strikingly. After lower, but stilllethal, doses of the enzyme, the hematocrit increases (readings as high as 80 have been obtained), and the percentage of hematin pigment in whole blood correspondingly increases. At least part of this loss of fluid from the blood is accounted for by increased water content of liver and kidney. After such doses of glucose oxidase, decreases were demonstrated in blood sugar and in liver glycogen.

Pretreatment of rats with phenobarbital activates the metabolism of acetophenetidin and of N -acetyl- p -aminophenol (NAPAP). The O -desalkylation of acetophenetidin and the conjugation of NAPAP with glucuronic acid are increased. These results were obtained by measuring the serum levels and by controlling the excretion of acetophenetidin, NAPAP and its conjugates in the urine. After administration of high doses of acetophenetidin in induced animals an increased concentration of methemoglobin was detected which indicates that the desacetylation and the N -hydroxylation are stimulated. Therefore pretreatment with phenobarbital in rats not only causes an enhanced detoxication of acetophenetidin, but also under high doses of acetophenetidin leads to an increase in its toxicity. The augmented conjugation of NAPAP after phenobarbital pretreatment, however, accelerates exclusively the detoxication.

The catalase inhibitor 3-amino-1,2,4-triazole was employed to detect hydrogen peroxide within erythrocytes; inhibition of catalase requires the presence of H2O2. H2O2 was detected during incubation of erythrocytes with the hemolytic drug acetylphenylhydrazine (APH). This result refutes a prior suggestion that APH does not generate H2O2, and supports the concept of H2O2 involvement during druginduced hemolysis of glucose 6-phosphate dehydrogenase-deficient cells. The following agents were more active than APH in generating H2O2: phenylhydrazine (100-fold), ascorbate (10-fold), and hydroquinone (3-fold). The reasons why a system based on methemoglobin formation in catalase-deficient cells failed to detect the H2O2 generated by APH are discussed.

The relationship between catalase and reducing equivalents in the red cell was studied by first exposing cells to an oxidative insult and then adding glucose as an energy source to facilitate recovery from the oxidative insult. Oxidative damage was produced by incubation of red cells with either 2 × 10 −6 M or 4 × 10 −5 M 1, 4-naphthoquinone-2-sulfonic acid, a vitamin K analog which generates Superoxide in the presence of oxyhemoglobin. After a 90 min incubation, cellular catalatic activity decreased to about 30 per cent of the original activity, NADH content was decreased to about 40 per cent, and NADPH content decreased to 10 per cent. At 90 min, d -glucose was added to a concentration of 5 mM. After a further 90 min incubation, red cells originally treated with the low concentration of quinone recovered catalatic activity to 70 per cent of the original activity, NADH to 60 per cent of the starting content, and NADPH content rose above 100 per cent. Red cells incubated with the higher quinone concentration did not recover catalatic activity or reduced nucleotide content. The data imply that catalase accumulates as the peroxidatic intermediate, Compound II, in the absence of a sufficient concentration of reducing equivalents. If oxidative damage is mild enough so that the glycolytic pathway and hexose monophosphate shunt can still restore an adequate level of reducing equivalents after the addition of glucose, then catalatic activity will be restored as the concentrations of ferricatalase and Compound I increase. The results indicate that catalase may function both peroxidatically and catalatically in the red cell.

Stimulation of the hexose monophosphate shunt by primaquine results from the oxidation of NADPH by primaquine. This conclusion was based on the observations that primaquine lowered cellular NADPH but not GSH and that, in red cells in which the GSH was unavailable for reaction, primaquine still stimulated the rate of the hexose monophosphate shunt. In a non-cellular system, primaquine interacted with NADPH, but not GSH, to produce H2O2. Stimulation of the hexose monophosphate shunt by primaquine does not primarily involve H2O2 accumulation since stimulation of the pathway by primaquine was also observed in red cells containing methemoglobin, a red cell preparation in which no H2O2 accumulates. Methemoglobin prevented the formation and/or accumulation of H2O2 in intact red cells incubated with primaquine as well as in a non-cellular system containing primaquine plus Fe2+-EDTA as an H2O2 source. Methemoglobin probably acts by scavenging reactive intermediates since oxyhemoglobin was formed from methemoglobin in the non-cellular experiments. In the red cell, primaquine stimulated glucose-dependent conversion of methemoglobin to oxyhemoglobin.

Uric acid has been postulated to be an important antioxidant and free radical scavenger in humans. Other purines, such as xanthine, that lack an 8-oxo group on the imidazole ring do not show antioxidant properties. For this reason, the antioxidative activities of 2-imidazolones and 2-imidazolthiones were compared to that of uric acid. 2-Imidazolthiones reacted with the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) at rates comparable to those of uric acid and other antioxidants. 2-Imidazolones also reacted with DPPH, although at a much slower rate than the 2-imidazolthiones. The 2-imidazolthiones protected oxyhemoglobin from oxidation to methemoglobin by sodium nitrite; the 2-imidazolones had little effect on the oxidation of oxyhemoglobin by nitrate. Most of the 2-imidazolthiones and 2-imidazolones protected both porcine and bovine erythrocytes from hemolysis by t-butyl hydroperoxide. Although 2-imidazolthiones were more reactive than 2-imidazolones in the assays using DPPH and the oxidation of oxyhemoglobin, both types of compounds may be useful as antioxidants in vivo.

Four distinct paramagnetic intermediates could be observed in the reaction between oxyhemoglobin and hydroxylamine using ESR spectroscopy. The radical species exhibited different stability properties thus different techniques were required for their detection. Two of them were identified as the hydronitroxide radical (NH 2 O) and the hemoglobin-nitric oxide complex (Hb 2+ −NO). The third one is a low-spin iron-(III)-complex, possibly the methemoglobin-hydroxylamine adduct. A fourth paramagnetic species was detected only in the absence of the iron chelator DETAPAC thus indicating that free iron ions were responsible for the formation of this intermediate. The same species was observed when a Fenton system was used to generate the radicals. This species was identified as being the Fe(NO) 2 X 2 complex described in the literature (X = inorganic anions such as OH − or PO 4 3− ). The identification of the radical intermediates detected in the hydroxylamine-induced methemoglobin formation contributes to a more detailed understanding of the reaction sequence.

The oxidative reactivities of four tryptophan metabolites in the kynurenine pathway were examined as a potential mechanism for their reported neurotoxicities and carcinogenicities. Neither quinolinic acid, a neurotoxin, nor its monocarboxylic analogue, picolinic acid, auto-oxidized over a wide pH range. However, 3-hydroxyanthranilic acid (3-HAT), a carcinogen, readily auto-oxidized and the reaction rate increased exponentially with increasing pH. 3-HAT auto-oxidation likely involves two steps: (1) auto-oxidation of 3-HAT to the semiquinoneimine (anthranilyl radical) which oxidizes to the quinoneimine, followed by (2) condensation and oxidation reactions to yield a second carcinogen, cinnabarinic acid. 3-HAT auto-oxidation to cinnabarinate required molecular oxygen and generated superoxide radicals and H2O2. Superoxide dismutase (SOD) accelerated 3-HAT auto-oxidation 4-fold, probably by preventing back reactions between Superoxide and either the anthranilyl radical or the quinoneimine formed during the initial step of auto-oxidation. Catalase did not accelerate 3-HAT auto-oxidation, but it did prevent destruction of cinnabarinate by H2O2. Interconversion between oxyhemoglobin and methemoglobin occurred during 3-HAT auto-oxidation, although neither form of hemoglobin altered rates of 3-HAT auto-oxidation. Mn2+, Mn3+ and Fe3+-EDTA did not directly catalyze cinnabarinate formation in the absence of O2, but they did accelerate cinnabarinate formation under aerobic conditions.

We have reported previously that the antimicrobial nitrofurantoin stimulates superoxide production and methemoglobin formation from HbO2 as an isolated hemeprotein and in hemolysates [M. Dershwitz and R. F. Novak, J. biol. Chem. 257, 75 (1982); M. Dershwitz and R. F. Novak, J. Pharmac. exp. Ther. 222, 430 (1982)]. The production of hydrogen peroxide and methemoglobin by nitrofurantoin has been determined in normal erythrocytes in vitro. Hydrogen peroxide production increased 5-fold during a 20-hr incubation in the presence of 840 microM nitrofurantoin, while methemoglobin content increased to over 20% of the total hemoglobin concentration of the cells. Consequent metabolic and morphologic alterations also occurred. Concomitant with nitrofurantoin-stimulated hydrogen peroxide production were time- and concentration-dependent decreases in cellular levels of GSH and ATP, as well as alterations in red cell morphology. Significant differences in GSH and ATP levels between control and nitrofurantoin-treated erythrocytes occurred after 12 hr and proceeded maximally from 18 to 21 hr. After a 21-hr incubation, 840 microM nitrofurantoin caused the cellular GSH and ATP levels to fall 65 and 75%, respectively, while controls exhibited only 29 and 43% decreases in ATP and GSH levels, respectively. Studies on the concentration dependence of such decreases demonstrated that the EC50 values for depletion of GSH and ATP were similar in blood obtained from an individual donor. The EC50 values varied from approximately 10 microM to 100 microM among the various donors whose blood was studied. Incubation of normal red cells with nitrofurantoin also resulted in an increased conversion of red cells to echinocytes as observed by scanning electron microscopy. These metabolic effects, coupled with increased oxidative stress via hydrogen peroxide generation, lend support to the mechanism for nitrofurantoin-induced hemolysis in erythrocytes compromised by certain enzyme deficiencies which result in low basal levels of GSH or diminished rates of GSH synthesis.