Noninvasive assessment of the relative roles of cerebral antioxidant enzymes by quantitation of pentose phosphate pathway activity.
Cerebral pentose phosphate pathway (PPP) plays a role in the biosynthesis of macromolecules, antioxidant defense and neurotransmitter metabolism. Studies on this potentially important pathway have been hampered by the inability to easily quantitate its activity, particularly in vivo. In this study we review the use of [1,6-13C2,6,6-2H2]glucose for measuring the relative activities of the PPP and glycolysis in a single incubation in cultured neurons and in vivo, when combined with microdialysis techniques. PPP activity in primary cerebrocortical cultures and in the caudate putamem of the rat in vivo was quantitated from data obtained by GC/MS analysis of released labeled lactate following metabolic degradation of [1,6-13C2,6,6-2H2]glucose. Exposure of cultures to H2O2 resulted in stimulation of PPP activity in a concentration-dependent fashion and subsequent cell death. Chelation of iron during H2O2 exposure exerted a protective effect thus implicating the participation of the Fenton reaction in mediating damage caused by the oxidative insult. Partial inhibition of glutathione peroxidase, but not catalase, was extremely toxic to the cultures reflecting the pivotal role of GPx in H2O2 detoxification. These results demonstrate the ability to dynamically monitor PPP activity and its response to oxidative challenges and should assist in facilitating our understanding of antioxidant pathways in the CNS.
Department of Radiology, University of Michigan, Ann Arbor 48109-0648, USA.
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Ben-Yoseph
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Boxer
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Ross
D000818:Animals; D002247:Carbon Isotopes; D002421:Caudate Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D003676:Deferoxamine / Q000494:pharmacology; D003903:Deuterium; D016291:Dizocilpine Maleate / Q000494:pharmacology; D005333:Fetus; D008401:Gas Chromatography-Mass Spectrometry; D005947:Glucose / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007501:Iron / Q000494:pharmacology; D007608:Kainic Acid / Q000494:pharmacology; D007700:Kinetics; D016202:N-Methylaspartate / Q000494:pharmacology; D009457:Neuroglia / Q000187:drug effects / Q000201:enzymology*; D009474:Neurons / Q000187:drug effects / Q000201:enzymology*; D010427:Pentose Phosphate Pathway; D011699:Putamen / Q000378:metabolism; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D002247:Carbon Isotopes; D011810:Quinoxalines; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D003903:Deuterium; D006861:Hydrogen Peroxide; D007501:Iron; D005947:Glucose; D003676:Deferoxamine; D007608:Kainic Acid