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Dynamic Measurements of Cerebral Pentose Phosphate Pathway Activity In Vivo Using [1,6‐ 13 C 2 ,6,6‐ 2 H 2 ] Glucose and Microdialysis
Author(s) -
BenYoseph Oded,
Camp Dianne M.,
Robinson Terry E.,
Ross Brian D.
Publication year - 1995
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1995.64031336.x
Subject(s) - isotopomers , microdialysis , pentose phosphate pathway , chemistry , in vivo , glycolysis , biochemistry , carbohydrate metabolism , metabolism , glucose 6 phosphate , enzyme , biology , extracellular , molecule , microbiology and biotechnology , organic chemistry
Cerebral pentose phosphate pathway (PPP) activity has been linked to NADPH‐dependent anabolic pathways, turnover of neurotransmitters, and protection from oxidative stress. Research on this potentially important pathway has been hampered, however, because measurement of regional cerebral PPP activity in vivo has not been possible. Our efforts to address this need focused on the use of a novel isotopically substituted glucose molecule, [1,6‐ 13 C 2 ,6,6‐ 2 H 2 ]glucose, in conjunction with microdialysis techniques, to measure cerebral PPP activity in vivo, in freely moving rats. Metabolism of [1,6‐ 13 C 2 ,6,6‐ 2 H 2 ]glucose through glycolysis produces [3‐ 13 C]lactate and [3‐ 13 C,3,3‐ 2 H 2 ]lactate, whereas metabolism through the PPP produces [3‐ 13 C,3,3‐ 2 H 2 ]lactate and unlabeled lactate. The ratios of these lactate isotopomers can be quantified using gas chromatography/mass spectrometry (GC/MS) for calculation of PPP activity, which is reported as the percentage of glucose metabolized to lactate that passed through the PPP. Following addition of [1,6‐ 13 C 2 ,6,6‐ 2 H 2 ]glucose to the perfusate, labeled lactate was easily detectable in dialysate using GC/MS. Basal forebrain and intracerebral 9L glioma PPP values (mean ± SD) were 3.5 ± 0.4 (n = 4) and 6.2 ± 0.9% (n = 4), respectively. Furthermore, PPP activity could be stimulated in vivo by addition of phenazine methosulfate, an artificial electron acceptor for NADPH, to the perfusion stream. These results show that the activity of the PPP can now be measured dynamically and regionally in the brains of conscious animals in vivo.