The role of metals in the enzymatic and nonenzymatic oxidation of epinephrine

The effects of transition metals on nonenzymatic and ceruloplasmin catalyzed epinephrine oxidation were investigated by studying rates of epinephrine oxidation in purified buffers and in the presence of metal chelating agents. We found that epinephrine does not “autoxidize” in sodium chloride solutions prepared with deionized water that was further purified by chromatography over Chelex 100 resin prior to use. Epinephrine was oxidized rapidly in sodium chloride prepared with tap water (1.20±0.12 nmoles/min) or in deionized water (0.40±0.80 nmoles/min), but this oxidation was prevented by the addition of Desferal, a potent metal chelating agent. Epinephrine oxidation was enhanced upon the addition of ceruloplasmin, and this oxidation rate could be slowed, but not eliminated, by the addition of Desferal. If epinephrine solutions were preincubated for 72 hours with Desferal prior to ceruloplasmin addition, however, no oxidation was observed. Epinephrine was shown to form colored complexes with both iron and copper at pH 7.0. The Fe(III)-epinephrine complex was much more stable than was the Cu(II)-epinephrine complex. Oxygen consumption studies of ceruloplasmin catalyzed epinephrine oxidation showed that copper was a better promoter of epinephrine oxidation than was iron, suggesting that ceruloplasmin-catalyzed epinephrine oxidation results from adventitious copper bound to the purified enzyme. In light of these results, the physiological relevance of ceruloplasmin catalyzed oxidation of biogenic amines may be minor.     

Possible role of hydroxyl radicals in the oxidation of dichloroacetonitrile by Fenton-like reaction

Dichloroacetonitrile (DCAN), is a member of haloacetonitrile group and detected in drinking water supplies as a by-product of chlorination process. The mechanism of DCAN-induced carcinogenesis is believed to be mediated by oxidative bioactivation of DCAN molecules. The present study was designed to investigate if reactive oxygen species (ROS), similar to that generated in biological systems, are capable of oxidative activation of DCAN. A model ROS generation system (Fenton-like reaction; Fe2+ and H2O2) that predominantly produces hydroxyl radical (OH*) was used. DCAN oxidation was monitored by the extent of cyanide (CN-) release. The results indicate that DCAN was markedly oxidized by this system, and the rate of oxidation was dependent on DCAN concentration. Four-fold increase in H2O2 concentration (50-200 mM) resulted in a 35-fold increase in CN- release. The rates of DACN oxidation in presence of various transition metals were in the following order; iron>copper>titanium. DCAN oxidation was enhanced significantly by the addition of vitamin C and sulfhydryl compounds such as glutathione, N-acetyl-L- cysteine, and dithiothreitol (10 mM) to 140, 130, 145 and 136% of control, respectively. Addition of H2O2 scavenger; catalase or iron chelator; desferrioxamine (DFO) resulted in a significant decrease in CN- release 47 and 41% of control, respectively. Addition of various concentrations of the free radical scavengers, DMSO, or mannitol, to the incubation mixtures caused a significant decrease in DCAN oxidation, 32 and 50% of control, respectively. Michaelis-Menten kinetic analysis of the rates of this reaction, with or without inhibitors, indicated that ROS mediated oxidation of DCAN was inhibited by catalase (Ki = 0.01 mM)>DFO (0.02 mM) > mannitol (0.09 mM) > DMSO (0.12 mM). In conclusion, our results indicate that DCAN is oxidized by a ROS-mediated mechanism. This mechanism may have an important role in DCAN bioactivation and DCAN-induced genotoxicity at target organs where multiple forms of ROS generating systems are abundant.     

The Effects of Different Buffers on the Oxidation of DNA by Thiols and Ferric Iron

Because buffers can act as metal ligands, they can effect several reactions necessary for DNA oxidation by ferric iron and thiols, such as iron reduction. Therefore, these reactions were studied in Hepes and phosphate buffers and unbuffered NaCl. Reduction of Fe³⁺ by dithiothreitol (DTT) and cysteine was observed in either Hepes or NaCl solutions, but not in phosphate buffer. Thiyl radicals were observed in Hepes, but there was much less thiyl radical production in the saline or phosphate solutions. Redox cycling between either DTT or cysteine and Fe³⁺ also resulted in dioxygen consumption in Hepes buffer. Reduction of Fe³⁺ and O₂ resulted in the formation of an oxidant capable of producing 8-hydroxy-2′-deoxyguanosine (8-OHdG) in calf-thymus DNA. The highest levels of 8-OHdG were detected when DTT or cysteine and Fe³⁺ were incubated in Hepes, while much less DNA oxidation was detected when the experiment was done in a saline solution, and almost no DNA oxidation occurred in the phosphate buffer. These results demonstrate that the use of different buffers can greatly affect the ability of thiols to promote iron-dependent oxidations. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 125–132, 1998     

Separation and identification of desferrioxamine and its iron chelating metabolites by high-performance liquid chromatography and fast atom bombardment mass spectrometry: choice of complexing agent and application to biological fluids

An HPLC-based method capable of separating desferrioxamine (DFO) and its iron chelating metabolites from uv-absorbing species present in biological fluids has been developed. This method relies on the use of nitrilotriacetic acid (NTA) as the complexing agent in the mobile phase, instead of EDTA, previously used in HPLC methods. The use of NTA ensures that iron contamination present in buffers and bound to the column does not interfere with analysis. The disadvantages of using EDTA are discussed. The identity of the iron chelating metabolites of DFO present in the urine of patients with beta-thalassemia major has been established using FAB mass spectrometry. The metabolism of DFO, reported in this study, takes place almost exclusively at the N-terminal region of the molecule and is in many respects similar to the degradation of the amino acid lysine. In addition, a metabolite which corresponds to N-hydroxylation of the terminal amino group has been identified.     

Inhibition of 2,3-dimethyl-1,4-naphthohydroquinone auto-oxidation by copper and by superoxide dismutase.

2,3-Dimethyl-1,4-naphthohydroquinone undergoes auto-oxidation to the corresponding quinone at pH 7.4, with stoichiometric consumption of oxygen and formation of hydrogen peroxide. In an unpurified buffer, the rate of oxidation was low, but it increased nearly 9-fold when trace metals were removed from the buffer by treatment with Chelex resin. A similar increase in rate was achieved by addition of DTPA or bathophenanthroline sulfonate to unpurified buffer, whereas EDTA and desferal were less effective. Addition of copper to purified buffer led to inhibition of oxidation, with a 50% decrease in rate being observed at a metal concentration of 7.1 nM, and it is likely that the low auto-oxidation rate recorded in unpurified buffer was due to copper contamination of the latter. The auto-oxidation of 2,3-dimethyl-1,4-naphthohydroquinone was exceptionally sensitive to inhibition by superoxide dismutase, with a concentration of only 4.5 ng/ml being sufficient for a 50% decrease in rate, and the inhibitory effect of copper may be due to the ability of this metal to catalyse the dismutation of superoxide. Previous studies have shown that the rates of auto-oxidation of 1,4-naphthohydroquinone and 2-methyl-1,4-naphthohydroquinone are influenced by copper contamination of buffer and the present study shows that this is also true for a di-substituted naphthohydroquinone. For accurate assessment of rates of naphthohydroquinone auto-oxidation, it is important that purified buffers or appropriate chelating agents, are employed.     

Comparison of iron-catalyzed DNA and lipid oxidation

Lipid and DNA oxidation catalyzed by iron(II) were compared in HEPES and phosphate buffers. Lipid peroxidation was examined in a sensitive liposome system constructed with a fluorescent probe that allowed us to examine the effects of both low and high iron concentrations. With liposomes made from synthetic 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine or from rat liver microsomal lipid, lipid peroxidation increased with iron concentration up to the range of 10--20 microM iron(II), but then rates decreased with further increases in iron concentration. This may be due to the limited amount of lipid peroxides available in liposomes for oxidation of iron(II) to generate equimolar iron(III), which is thought to be important for the initation of lipid peroxidation. Addition of hydrogen peroxide to incubations with 1--10 microM iron(II) decreased rates of lipid peroxidation, whereas addition of hydrogen peroxide to incubations with higher iron concentrations increased rates of lipid peroxidation. Thus, in this liposome system, sufficient peroxide from either within the lipid or from exogenous sources must be present to generate equimolar iron(II) and iron(III). With iron-catalyzed DNA oxidation, hydrogen peroxide always stimulated product formation. Phosphate buffer, which chelates iron but still allows for generation of hydroxyl radicals, inhibited lipid peroxidation but not DNA oxidation. HEPES buffer, which scavenges hydroxyl radicals, inhibited DNA oxidation, whereas lipid peroxidation was unaffected since presumably iron(II) and iron(III) were still available for reaction with liposomes in HEPES buffer.     

Ascorbate Autoxidation in the Presence of Iron and Copper Chelates

Chelates can inhibit the iron- and copper-catalyzed autoxidation of ascorbate at pH 7.0. Diethylenetri-aminepentaacetic acid (DTPA or DETAPAC) and Desferal (deferoximane mesylate) slow the iron-catalyzed oxidation of ascorbate as effectively as reducing the trace levels of contaminating iron in buffers with Chelex resin. DETAPAC, EDTA and HEDTA (N-(2-hydroxyethyl)-ethylenediaminetriacetic acid) are effective at slowing the copper-catalyzed autoxidation of ascorbate while Desferal is ineffective. The ability to inhibit ascorbate autoxidation appears to parallel the rate of the reaction of superoxide with the iron chelate.     

Ascorbate Autoxidation in the Presence of Iron and Copper Chelates

Chelates can inhibit the iron- and copper-catalyzed autoxidation of ascorbate at pH 7.0. Diethylenetri-aminepentaacetic acid (DTPA or DETAPAC) and Desferal (deferoximane mesylate) slow the iron-catalyzed oxidation of ascorbate as effectively as reducing the trace levels of contaminating iron in buffers with Chelex resin. DETAPAC, EDTA and HEDTA (N-(2-hydroxyethyl)-ethylenediaminetriacetic acid) are effective at slowing the copper-catalyzed autoxidation of ascorbate while Desferal is ineffective. The ability to inhibit ascorbate autoxidation appears to parallel the rate of the reaction of superoxide with the iron chelate.     

Iron Autoxidation in Mops and Hepes Buffers

Iron autoxidation in Mops and Hepes buffers is characterized by a lag phase that becomes shorter with increasing FeCl₂ concentration and pH. During iron oxidation in these buffers a yellow colour develops in the solution. When the reaction is conducted in the presence of nitro blue tetrazolium (NBT), blue formazan is formed. Of the many OH' scavengers tested, mannitol and sorbitol are most effective in inhibiting Fe²⁺ oxidation, yellow colour development and NBT reduction. Some inhibition was also noted with catalase. The iron product of the oxidative reaction differs from Fe³⁺ in its absorption spectrum and its low reactivity with thiocyanate. Similar results are obtained when iron autoxidation is studied in unbuffered solutions brought to alkaline pH with NaOH. In phosphate buffer, no lag phase is evident and the absorption spectrum of the final solution is identical to that of Fe³⁺ in this buffer. The iron product reacts immediately with thiocyanate. When iron oxidation is conducted in the presence of NBT the formation of formazan is almost undetectable. Of the many compounds tested only catalase inhibits iron autoxidation in this buffer. The sequence of reactions leading to iron autoxidation in Good-type buffers¹ thus resembles that occurring in unbuffered solutions brought to alkaline pH with NaOH and greatly differs from that occurring in phosphate buffer. These results are in agreement with the observation that these buffers have very low affinity for iron.¹ The data presented define experimental conditions where Fe²⁺ is substantially stable for a considerable length of time in Mops buffer.     

Endosomal and lysosomal effects of desferrioxamine: protection of HeLa cells from hydrogen peroxide-induced DNA damage and induction of cell-cycle arrest

The role of endosomal/lysosomal redox-active iron in H2O2-induced nuclear DNA damage as well as in cell proliferation was examined using the iron chelator desferrioxamine (DFO). Transient transfections of HeLa cells with vectors encoding dominant proteins involved in the regulation of various routes of endocytosis (dynamin and Rab5) were used to show that DFO (a potent and rather specific iron chelator) enters cells by fluid-phase endocytosis and exerts its effects by chelating redox-active iron present in the endosomal/lysosomal compartment. Endocytosed DFO effectively protected cells against H2O2-induced DNA damage, indicating the importance of endosomal/lysosomal redox-active iron in these processes. Moreover, exposure of cells to DFO in a range of concentrations (0.1 to 100 microM) inhibited cell proliferation in a fluid-phase endocytosis-dependent manner. Flow cytometric analysis of cells exposed to 100 microM DFO for 24 h showed that the cell cycle was transiently interrupted at the G2/M phase, while treatment for 48 h led to permanent cell arrest. Collectively, the above results clearly indicate that DFO has to be endocytosed by the fluid-phase pathway to protect cells against H2O2-induced DNA damage. Moreover, chelation of iron in the endosomal/lysosomal cell compartment leads to cell cycle interruption, indicating that all cellular labile iron is propagated through this compartment before its anabolic use is possible.     

Aluminum effect on the activity of superoxide dismutase and of other antioxygenic enzymes in vitro

The effect of Al on superoxide dismutase (SOD) and on other antioxygenic enzymes: horseradish peroxidase, catalase, and glutathione peroxidase, has been investigated in vitro. In the case of SOD, the effect of metal chelators (EDTA and deferoxamine) and a possible synergistic effect with iron salts have also been tested using the pyrogallol assay. There is no significant inhibitory effect of Al on the activity of any of the above-mentioned enzymes. Noticeable increases in SOD activity were observed when metal chelators were added to the medium, but not when high concentrations of Al were present too, in the case of deferoxamine (DFO). The former fact seems to be a consequence of the chelation of transition metal ions that catalyze pyrogallol autoxidation by a mechanism not inhibitable by SOD, interfering in its action, which may account for part of the DFO antioxidant effect observed in vivo. The latter phenomenon could be owing to a saturation of the chelating capacity of DFO by an excess of Al present in the medium, which should bring the system back to the interfering conditions explained above. It can be concluded that Al, either in the presence or in the absence of iron salts, does not inhibit SOD activity in vitro. Moreover, no significant binding of Al to SOD was demonstrated, and the amounts of its metal constituents, Cu and Zn, were not affected by preincubation of the enzyme with Al. The effect of the different compounds tested on the rate of autoxidation of the indicating scavenger, pyrogallol, and a suitable hypothesis on their role in the oxidation process are also discussed.     

Hemoglobin: A mechanism for the generation of hydroxyl radicals

Oxyhemoglobin (HbO₂) reduces Fe(III) NTA aerobically to become methemoglobin (metHb) and Fe(II)NTA. These conditions are favorable for the generation via Fenton chemistry of the hydroxyl radical that was measured by HPLC using salicylate as a probe. The levels of hydroxyl radicals generated are a function of both the percent metHb formed and the chemical nature of the buffer. The rates of formation of both metHb and hydroxyl radicals were dependent upon the concentration of Fe(III)NTA. Of the buffers tested, HEPES was the most effective scavenger of hydroxyl radicals while the other buffers scavenged in the order: HEPES > Tris > MOPS > NaCl ≈ unbuffered. The addition of catalase to remove H₂0₂ or bathophenanthroline to chelate Fe(II) inhibited virtually all hydroxyl radical formation. Carbonyl formation from free radical oxidation of amino acids was found to be 0.1 mol/mol of hemoglobin. These experiments demonstrate the ability of hemoglobin to participate directly in the generation of hydroxyl radicals mediated by redox metals, and provide insight into potential oxidative damage from metals released into the blood during some pathologic disorders including iron overload.     

In the absence of catalytic metals ascorbate does not autoxidize at pH 7: ascorbate as a test for catalytic metals

Trace amounts of adventitious transition metals in buffer solutions can serve as catalysts for many oxidative processes. To fully understand what role these metals may play it is necessary that buffer solutions be 'catalytic metal free'. We demonstrate here that ascorbate can be used in a quick and easy test to determine if near-neutral buffer solutions are indeed 'catalytic metal free'. In buffers which have been rendered free of catalytic metals we have found that ascorbate is quite stable, even at pH 7. The first-order rate constant for the loss of ascorbate in an air-saturated catalytic metal free solution is less than 6 X 10(-7) s-1 at pH 7.0. This upper limit appears to be set by the inability to completely eliminate catalytic metal contamination of solutions and glassware. We conclude that in the absence of catalytic metals, ascorbate is stable at pH 7.     

Hydroxyl radical is not a product of the reaction of xanthine oxidase and xanthine. The confounding problem of adventitious iron bound to xanthine oxidase

The reaction of xanthine and xanthine oxidase generates superoxide and hydrogen peroxide. In contrast to earlier works, recent spin trapping data (Kuppusamy, P., and Zweier, J.L. (1989) J. Biol. Chem. 264, 9880-9884) suggested that hydroxyl radical may also be a product of this reaction. Determining if hydroxyl radical results directly from the xanthine/xanthine oxidase reaction is important for 1) interpreting experimental data in which this reaction is used as a model of oxidant stress, and 2) understanding the pathogenesis of ischemia/reperfusion injury. Consequently, we evaluated the conditions required for hydroxyl radical generation during the oxidation of xanthine by xanthine oxidase. Following the addition of some, but not all, commercial preparations of xanthine oxidase to a mixture of xanthine, deferoxamine, and either 5,5-dimethyl-1-pyrroline-N-oxide or a combination of alpha-phenyl-N-tert-butyl-nitrone and dimethyl sulfoxide, hydroxyl radical-derived spin adducts were detected. With other preparations, no evidence of hydroxyl radical formation was noted. Xanthine oxidase preparations that generated hydroxyl radical had greater iron associated with them, suggesting that adventitious iron was a possible contributing factor. Consistent with this hypothesis, addition of H2O2, in the absence of xanthine, to "high iron" xanthine oxidase preparations generated hydroxyl radical. Substitution of a different iron chelator, diethylenetriaminepentaacetic acid for deferoxamine, or preincubation of high iron xanthine oxidase preparations with chelating resin, or overnight dialysis of the enzyme against deferoxamine decreased or eliminated hydroxyl radical generation without altering the rate of superoxide production. Therefore, hydroxyl radical does not appear to be a product of the oxidation of xanthine by xanthine oxidase. However, commercial xanthine oxidase preparations may contain adventitious iron bound to the enzyme, which can catalyze hydroxyl radical formation from hydrogen peroxide.     

Quantification of desferrioxamine and its iron chelating metabolites by high-performance liquid chromatography and simultaneous ultraviolet-visible/radioactive detection.

An HPLC-based method for quantification of desferrioxamine (DFO) and its iron chelating metabolites in plasma has been developed. This assay overcomes stability problems associated with DFO by the addition of radioactive iron to convert unbound drug and metabolites to radio-iron-bound species. A dual detection system utilizing uv-vis absorption and radioactive (beta-particle) detector was used to quantify total and radio-iron-bound species. The use of octadecyl silanol solid phase extraction cartridges permits concentration of samples and allows accurate quantification of drug and metabolites down to 0.1 nmol/ml.     

Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal.

The difficulties associated with the growth of Legionella species in common laboratory media may be due to the sensitivity of these organisms to low levels of hydrogen peroxide and superoxide radicals. Exposure of yeast extract (YE) broth to fluorescent light generated superoxide radicals (3 microM/h) and hydrogen peroxide (16 microM/h). Autoclaved YE medium was more prone to photochemical oxidation than YE medium sterilized by filtration. Activated charcoals and, to a lesser extent, graphite, but not starch, prevented photochemical oxidation of YE medium, decomposed hydrogen peroxide and superoxide radicals, and prevented light-accelerated autooxidation of cysteine. Also, suspensions of charcoal in phosphate buffer and in charcoal yeast extract medium readily decomposed exogenous peroxide (17 and 23 nmol/ml per min, respectively). Combinations of bovine superoxide dismutase and catalase also decreased the rate of photooxidation of YE medium. Medium protected from light did not accumulate appreciable levels of hydrogen peroxide, and autoclaved YE medium protected from light supported good growth of Legionella micdadei. Various species of Legionella (10(4) cells per ml) exhibited sensitivity to relatively low levels of hydrogen peroxide (26.5 microM) in challenge experiments. The level of hydrogen peroxide that accumulated in YE medium over a period of several hours (greater than 50 microM) was in excess of the level tolerated by Legionella pneumophila, which contained no measurable catalase activity. Strains of L. micdadei, Legionella dumoffi, and Legionella bozmanii contained this enzyme, but the presence of catalase did not appear to confer appreciable tolerance to exogenously generated hydrogen peroxide.     

Preparation of metal ion buffers for biological experimentation: a methods approach with emphasis on iron and zinc

Transition metal ions are a challenge to study in physiology because of problems associated with solubility, oxidation, binding, and attaining appropriate free activities in solution. This review discusses these problems and potential ways of accommodating them. Special attention is given to iron and zinc ions, but many of the concepts can be applied for studying other transition metals. Selection of reagents appropriate for metal work (including water, salts, noncomplexing pH buffers) is briefly discussed. Calculation of the solubility product (K(sp)) for common iron and zinc precipitates is covered, as well as techniques used to solubilize Fe(3+) with organic chelates. Factors that affect Fe(2+) oxidation are mentioned, and the use of ascorbate as a reducing agent is considered. Measurement of the rate of Fe(2+) oxidation (or Fe(3+) reduction) with the Fe(2+) chromophores ferrozine and BPS is also discussed. Generation of a free metal ion activity through use of metal buffers (chelators) is discussed. Theoretical problems associated with this technique are explored, and selected shareware metal ion buffer calculators are described. Finally, techniques for measuring and minimizing nonspecific binding of iron and zinc ions to biological membranes are considered.     

Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal

The difficulties associated with the growth of Legionella species in common laboratory media may be due to the sensitivity of these organisms to low levels of hydrogen peroxide and superoxide radicals. Exposure of yeast extract (YE) broth to fluorescent light generated superoxide radicals (3 microM/h) and hydrogen peroxide (16 microM/h). Autoclaved YE medium was more prone to photochemical oxidation than YE medium sterilized by filtration. Activated charcoals and, to a lesser extent, graphite, but not starch, prevented photochemical oxidation of YE medium, decomposed hydrogen peroxide and superoxide radicals, and prevented light-accelerated autooxidation of cysteine. Also, suspensions of charcoal in phosphate buffer and in charcoal yeast extract medium readily decomposed exogenous peroxide (17 and 23 nmol/ml per min, respectively). Combinations of bovine superoxide dismutase and catalase also decreased the rate of photooxidation of YE medium. Medium protected from light did not accumulate appreciable levels of hydrogen peroxide, and autoclaved YE medium protected from light supported good growth of Legionella micdadei. Various species of Legionella (10(4) cells per ml) exhibited sensitivity to relatively low levels of hydrogen peroxide (26.5 microM) in challenge experiments. The level of hydrogen peroxide that accumulated in YE medium over a period of several hours (greater than 50 microM) was in excess of the level tolerated by Legionella pneumophila, which contained no measurable catalase activity. Strains of L. micdadei, Legionella dumoffi, and Legionella bozmanii contained this enzyme, but the presence of catalase did not appear to confer appreciable tolerance to exogenously generated hydrogen peroxide.     

D-Penicillamine: analysis of the mechanism of copper-catalyzed hydrogen peroxide generation

Recent studies have suggested that the inhibition of lymphocyte mitogenesis by D-penicillamine in the presence of copper could be mediated by the formation and action of hydrogen peroxide. To explore this possibility further, we first sought evidence of H2O2 generation by D-penicillamine in a cell-free system by a) measurement of copper-catalyzed D-penicillamine oxidation and the requirement for oxygen in this process; b) direct measurement of H2O2 formation during D-penicillamine oxidation by the peroxidase-mediated oxidation of fluorescent scopoletin; and c) evaluation of the possible synthesis of O2- during D-penicillamine oxidation. The addition of copper to D-penicillamine in physiologic buffer catalyzed D-penicillamine oxidation in a dose-dependent fashion. D-penicillamine oxidation was accompanied by O2 consumption with a molar ratio of approximately 2:1, but did not occur under anaerobic conditions. Furthermore, D-penicillamine oxidation resulted in the formation of amounts of H2O2 stoichiometrically equivalent to oxygen consumption (i.e., 1:1). Copper-catalyzed D-penicillamine oxidation caused reduction of nitroblue tetrazolium in a reaction blocked by superoxide dismutase, suggesting the formation of O2-. Additional studies confirmed that D-penicillamine inhibited PHA-induced mitogenesis of lymphocytes in the presence of copper, and that catalase protected the cells from this action. Furthermore, when polymorphonuclear leukocytes were incubated with D-penicillamine plus copper, hexose monophosphate shunt activity increased up to threefold with abrogation of this stimulation by catalase. None of the effects of D-penicillamine plus copper on cells were diminished by hydroxyl radical scavengers mannitol or benzoate. These results are consistent with oxygen-dependent copper-catalyzed oxidation of D-penicillamine in aqueous solutions leading to the formation of O2- and H2O2. H2O2 produced by this reaction can inhibit lymphocyte mitogenesis and stimulate neutrophil hexose monophosphate shunt activity in vitro and may be relevant to the therapeutic effects of D-penicillamine in vivo.     

Desferrioxamine protects human red blood cells from hemin-induced hemolysis

Hemin binding to red cell membranes, its effect on red cell hemolysis, and it interaction with desferrioxamine (DFO) in these processes were investigated. DFO interacted with hemin via the iron moiety. Blockage of the binding groups in DFO prevented interaction of DFO with hemin, implying the importance of the hydroxamic acid groups in DFO-hemin interactions. Since hemolysis is a result of hemin association with the membrane components, its binding in the presence and absence of DFO was studied. DFO strongly inhibited hemin-induced lysis in a concentration-dependent manner. With 50 microM hemin, 1 mM DFO completely inhibited lysis. Preincubation of ghost membranes with DFO (1 mM) inhibited binding of hemin (50 microM) to membranes by 42%. After ghost membranes were preincubated with hemin (50 microM), the addition of DFO (1 mM) removed 20% of the membrane-bound hemin. It is suggested that DFO may have an important role in alleviating the hemin-induced deleterious effects on the red cell membrane, especially in hemolytic anemias associated with unstable, autoxidized hemoglobins.