The quantitative determination of the in vivo dephosphorylation of glucose 6-phosphate in rat brain.

An isotopic method was developed to measure the in vivo rate of dephosphorylation of glucose 6-phosphate in brains of conscious rats. The rats were surgically prepared 20-24 h before the experiment by: 1) cannulation of the left carotid artery, 2) ligation of the left external carotid artery, and 3) ligation of the right internal carotid artery. A mixture of [2-3H,U-'4C]glucose was injected into the left internal carotid artery and the change in the 3H/'4C ratio of cerebral glucose was examined in freeze-blown brains at various times following injection. The 3H/14C ratio in cerebral glucose decreased approximately 35% in the 5 min following injection. During the same period the isotopic ratio in blood glucose decreased less than 5%. This indicates that the decrease in 3H/'4C ratio in cerebral gluocse was caused by the dephosphorylation of glucose 6phosphate in cerebral tissues, not by uptake of the recirculated labeled glucose by brain. The rate of dephosphorylation of glucose 6-phosphate was calculated by subtracting the rate of phosphorylation of [U-'"C] glucose from that of [2-3H]glucose. The rate of phosphorylation (F) of labeled glucose was calculated as follows. F = (disintegration/min in metabolic products of glucose/g of tissue)/(Specific activity of glucose (dpm*pmol")*time (min)) The extrapolated rate of flux through glycolysis measured with ~-'4C]glucose was 0.86 pmol-min"* g-I. The rate of dephosphorylation was estimated as 0.30 pmol-min"-g-';xabout 35% of the rate of glucose utilization. The 3H/ C ratio in cerebral glucose from rats injected with 16-3H,U-'4C]glucose did not show significant changes in 10 min, indicating further that the dephosphorylation of glucose 6-phosphate observed in the experiments with [2-3H,U-'4C] glucose was not the result of recirculation of label into brain from peripheral tissues or the reversal of the entire glycolytic pathway.