TY - JOUR
T1 - Oxidative Decarboxylation of Benzoate to Carbon Dioxide by Rat Liver Microsomes
T2 - A Probe for Oxygen Radical Production during Microsomal Electron Transfer
AU - Winston, Gary W.
AU - Cederbaum, Arthur I.
PY - 1982/8
Y1 - 1982/8
N2 - The oxidative decarboxylation of [7-14C] benzoate has been used by others to evaluate -OH production by phagocytes and during the xanthine-xanthine oxidase reaction. The current report concerns the use of benzoate as a probe for the detection of oxy radicals generated by another biological system, NADPH-dependent, microsomal electron transfer. 14CO2 was produced from [7-14C]benzoate by rat liver microsomes in a Chelex-treated incubation medium. The reaction was dependent upon an NADPH-generating system and intact microsomes. 14CO2 was augmented nearly 10-fold in the presence of azide, an inhibitor of catalase, suggesting that H2O2 may serve as a precursor of -OH. External addition of H2O2 also increased benzoate decarboxylation. The -OH scavengers mannitol, dimethyl sulfoxide, 2-oxo-4-(methylthio) butyric acid, and ethanol inhibited 14CO2 production. Kinetic studies suggested that the scavengers compete with benzoate for a common intermediate or site. Iron-ethylenediaminetetraacetic acid (EDTA), which catalyzes the decomposition of H202 resulting in the generation of -OH (Fen-ton-type reaction), stimulated 14CO2 production in a dosedependent manner. Metyrapone and SKF-525A, typical inhibitors of mixed-function oxidase activity, did not significantly affect the decarboxylation of benzoate. Organic hydroperoxides such as cumene or terf-butyl hydroperoxide, which are capable of catalyzing the metabolism of certain drugs in the absence of NADPH, did not catalyze benzoate decarboxylation. These results disassociate the overall metabolism of benzoate from typical substrates of the mixed-function oxidase system and suggest that discrete pathways for microsomal metabolism of drugs and -OH scavengers exist. The autoxidation of ascorbate by Fe3+ readily promoted benzoate decarboxylation in a manner consistent with a role for -OH. These data suggest that 14CO2 production from [7-14C]- benzoate serves as a sensitive, simple, and efficacious probe for the production of an oxidative radical or radicals generated during microsomal electron transport. This oxidant appears to resemble -OH in its oxidizing properties.
AB - The oxidative decarboxylation of [7-14C] benzoate has been used by others to evaluate -OH production by phagocytes and during the xanthine-xanthine oxidase reaction. The current report concerns the use of benzoate as a probe for the detection of oxy radicals generated by another biological system, NADPH-dependent, microsomal electron transfer. 14CO2 was produced from [7-14C]benzoate by rat liver microsomes in a Chelex-treated incubation medium. The reaction was dependent upon an NADPH-generating system and intact microsomes. 14CO2 was augmented nearly 10-fold in the presence of azide, an inhibitor of catalase, suggesting that H2O2 may serve as a precursor of -OH. External addition of H2O2 also increased benzoate decarboxylation. The -OH scavengers mannitol, dimethyl sulfoxide, 2-oxo-4-(methylthio) butyric acid, and ethanol inhibited 14CO2 production. Kinetic studies suggested that the scavengers compete with benzoate for a common intermediate or site. Iron-ethylenediaminetetraacetic acid (EDTA), which catalyzes the decomposition of H202 resulting in the generation of -OH (Fen-ton-type reaction), stimulated 14CO2 production in a dosedependent manner. Metyrapone and SKF-525A, typical inhibitors of mixed-function oxidase activity, did not significantly affect the decarboxylation of benzoate. Organic hydroperoxides such as cumene or terf-butyl hydroperoxide, which are capable of catalyzing the metabolism of certain drugs in the absence of NADPH, did not catalyze benzoate decarboxylation. These results disassociate the overall metabolism of benzoate from typical substrates of the mixed-function oxidase system and suggest that discrete pathways for microsomal metabolism of drugs and -OH scavengers exist. The autoxidation of ascorbate by Fe3+ readily promoted benzoate decarboxylation in a manner consistent with a role for -OH. These data suggest that 14CO2 production from [7-14C]- benzoate serves as a sensitive, simple, and efficacious probe for the production of an oxidative radical or radicals generated during microsomal electron transport. This oxidant appears to resemble -OH in its oxidizing properties.
UR - http://www.scopus.com/inward/record.url?scp=0020494555&partnerID=8YFLogxK
U2 - 10.1021/bi00261a013
DO - 10.1021/bi00261a013
M3 - Article
C2 - 6289875
AN - SCOPUS:0020494555
SN - 0006-2960
VL - 21
SP - 4265
EP - 4270
JO - Biochemistry
JF - Biochemistry
IS - 18
ER -