Spin trapping evidence for free radical oxidants of aminopyrine in the metmyoglobin—cumene hydroperoxide system

Griffin, Brenda Walker; Ting, Patrick L.

doi:10.1016/0014-5793(78)80216-6

Cited by 25 publications

(4 citation statements)

References 18 publications

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“…Hence DPA was observed to be a highly effective lipid-peroxidation inhibitor in the red cell and less so in ghosts. Consistent with our results suggesting haemoglobin-promoted decomposition of t-butyl hydroperoxide to free radicals are the results of Griffin & Ting (1978), where in the reaction of metmyoglobin with cumene hydroperoxide oxidative destruction of the haem groups and production of the cumyloxyl radical is observed.…”

Section: Discussionsupporting

confidence: 80%

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Trotta

Sullivan

Stern

1983

Biochemical Journal

149

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Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin-and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmonooxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the p...

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Section: Discussionsupporting

confidence: 80%

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Trotta

Sullivan

Stern

1983

Biochemical Journal

149

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“…Thus, under the conditions of our experiments, it is reasonable to project that at CHP or BHP concentrations of less than 4 mM, all organic hydroperoxide would have been consumed in ~1 h and that protein (Hb) damage occurred rapidly via generation of alkoxyl or alkyl radicals after reaction with Hb. Consistent with this mechanism for protein damage is the observation that reaction of CHP with metmyoglobin results in destruction of heme and production of the cumyloxyl radical (23). Thus, hydroperoxide-derived free radicals readily react with heme proteins, resulting in protein damage (24).…”

Section: Discussionmentioning

confidence: 82%

Differential effects of organic hydroperoxides and hydrogen peroxide on proteolysis in human erythrocytes

Runge‐Morris¹,

Frank²,

Novak³

1989

Chem. Res. Toxicol.

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The effects of tert-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide on proteolysis in human red blood cells have been examined. The organic hydroperoxides effectively stimulated the rate of protein degradation in red cells and in hemolysate; in contrast, H2O2 addition was without significant effect in either system. tert-Butyl hydroperoxide or cumene hydroperoxide (8 mM) increased the rate of protein degradation in red cells 2.3- and 4-fold, respectively, relative to control as monitored by tyrosine release. In hemolysate, tert-butyl hydroperoxide and cumene hydroperoxide, present at 8 mM, produced a 2- and 3-fold increase in the rate of protein degradation, respectively, as compared to controls. Hydroperoxide-stimulated proteolysis in red cells or in hemolysate was concentration-dependent and reached saturation at 8 mM hydroperoxide. The reaction was linear for 2 h after which a plateau was reached. In contrast to the results observed for the organic hydroperoxides, H2O2 (100 or 200 mM) addition either alone or in the presence of the catalase inhibitor 3-amino-1,2,4-triazole (50-200 mM), failed to stimulate proteolysis. N-Acetylcysteine (20 mM) and dimethylthiourea (50 mM) inhibited the rate of hydroperoxide-stimulated proteolysis in red cells by approximately 50 and approximately 35%, respectively, and in hemolysate by 25 and 40%, respectively. The hydroxyl radical scavengers methyl sulfoxide (50 mM) or dimethylfuran (50 mM), metal ion chelators, or spin traps failed to decrease significantly the rate of organic hydroperoxide stimulated proteolysis. In addition, inhibitors of the calpain/procalpain system in red cell or hemolysate incubations challenged by organic hydroperoxide were without significant effect on the rate of proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…However, when 2-methyl-2-nitrosopropane (MNP) was present, the reaction produced an epr signal, Fig. 3, which contained two nitroxide radicals, assigned to spin adducts of MNP with a methyl radical and a butyl radical (14). The latter nitroxide has been shown to arise from light-induced decomposition of the spin trap (22), and was, accordingly, observed in all control experiments.…”

mentioning

confidence: 83%

“…All other reagents were the highest quality commercially available. The experimental techniques employed for the epr experiments, which made use of a Varian E-4 spectrometer interfaced to a Digital Equipment Corporation PDP 11/05 computer, have been described in detail elsewhere (13,14). Details of the high-performance liquid chromatographic analysis of the products of decomposition of cumene hydroperoxide have been published (14).…”

Section: Methodsmentioning

confidence: 99%

Use of spin traps to elucidate radical mechanisms of oxidations by hydroperoxides catalyzed by hemeproteins

Griffin¹

1982

Can. J. Chem.

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). The use of the spin traps nitrosobenzene and 2-methyl-2-nitrosopropane has established that metmyoglobin and liver microsomal cytochrome P-450 initiate a radical decomposition of cumene hydroperoxide. With metmyoglobin and the alkyl nitroso compound, the only radical product of cumene hydroperoxide trapped was the methyl radical formed by P scission of the cumyloxy radical.With both hemeprotein initiators, nitrosobenzene trapped only the cumyl radical, considered to be a decomposition product of the unstable spin adduct phenylcumyloxynitroxide. Support for this proposal includes: (1) previous spin trapping studies of the chemical decomposition of cumene hydroperoxide; and (2) significant inhibition by nitrosobenzene of the one-electron oxidation of aminopyrine and the autoxidation of unsaturated membrane lipids resultingfrom addition of the hydroperoxide to liver microsomes. Aminopyrine altered the epr signal amplitudes of the spin adducts produced with both nitroso compounds, indicative of oxidation of aminopyrine by the methyl radical and reduction of cumene hydroperoxide by the aminopyrine radical. The participation of hydroperoxide-derived radicals in the low peroxidatic activities of certain hemeproteins is quite distinct from the catalytic function of the true hemeprotein peroxidases, which bring about an efficient two-electron reduction of specific hydroperoxides.

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Spin trapping evidence for free radical oxidants of aminopyrine in the metmyoglobin—cumene hydroperoxide system

Abstract: Preliminary accounts of this work were presented at the American Chemical Society Southeast—Southwest Regional Meeting, October 29–31, 1975, Memphis, TN and at the American Chemical Society Southwest Regional Meeting, December 5–7, 1977, Little Rock, AR

Cited by 25 publications

References 18 publications

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Differential effects of organic hydroperoxides and hydrogen peroxide on proteolysis in human erythrocytes

Use of spin traps to elucidate radical mechanisms of oxidations by hydroperoxides catalyzed by hemeproteins

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