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 biosynthe sis of macromolecules, antioxidant defense and neurotransmitter metabo lism. Studies on this potentially important pathway have been hampered by the inability to easily quantitate its activity, particularly in v ivo. in this study we review the use of [1,6-C-13(2),6,6-H-2(2)]glucos e 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 cul tures and in the caudate putamem of the rat in vivo was quantitated fr om data obtained by CC/MS analysis of released labeled lactate followi ng metabolic degradation of [1,6-C-13(2),6,6-H-2(2)]glucose. Exposure of cultures to H2O2 resulted in stimulation of PPP activity in a conce ntration-dependent fashion and subsequent cell death. Chelation of iro n during H2O2 exposure exerted a protective effect thus implicating th e participation of the Fenton reaction in mediating damage caused by t he oxidative insult. Partial inhibition of glutathione peroxidase, but not catalase, was extremely toxic to the cultures reflecting the pivo tal role of GPx in H2O2 detoxification. These results demonstrate the ability to dynamically monitor PPP activity and its response to oxidat ive challenges and should assist in facilitating our understanding of antioxidant pathways in the CNS.