DYNAMIC MEASUREMENTS OF CEREBRAL PENTOSE-PHOSPHATE PATHWAY ACTIVITY IN-VIVO USING [1,6-C-13(2),6,6-H-2(2)]GLUCOSE AND MICRODIALYSIS

Cerebral pentose phosphate pathway (PPP) activity has been linked to N ADPH-dependent anabolic pathways, turnover of neurotransmitters, and p rotection from oxidative stress. Research on this potentially importan t pathway has been hampered, however, because measurement of regional cerebral PPP activity in vivo has not been possible. Our efforts to ad dress this need focused on the use of a novel isotopically substituted glucose molecule, [1,6-C-13(2),6,6-H-2(2)] glucose, in conjunction wi th microdialysis techniques, to measure cerebral PPP activity in vivo, in freely moving rats. Metabolism of [1,6-C-13(2),6,6-H-2(2)] glucose through glycolysis produces [3-C-13] lactate and [3-C-13,3,3-H-2(2)] lactate, whereas metabolism through the PPP produces [3-C-13,3,3-H-2(2 )] lactate and unlabeled lactate. The ratios of these lactate isotopom ers can be quantified using gas chromatography/mass spectrometry (GC/M S) for calculation of PPP activity, which is reported as the percentag e of glucose metabolized to lactate that passed through the PPP. Follo wing addition of [1,6-C-13(2),6,6-H-2(2)] glucose to the perfusate, la beled lactate was easily detectable in dialysate using GC/MS. Basal fo rebrain and intracerebral 9L glioma PPP values (mean +/- SD) were 3.5 +/- 0.4 (n = 4) and 6.2 +/- 0.9% (n = 4), respectively. Furthermore, P PP activity could be stimulated in vivo by addition of phenazine metho sulfate, an artificial electron acceptor for NADPH, to the perfusion s tream. These results show that the activity of the PPP can now be meas ured dynamically and regionally in the brains of conscious animals in vivo.