IN-SITU MEASUREMENT OF GLUTAMATE CONCENTRATIONS IN THE PERIPORTAL, INTERMEDIATE, AND PERICENTRAL ZONES OF RAT-LIVER

We developed a quantitative histochemical assay for measurement of loc al glutamate concentrations in cryostat sections of rat liver. Deamina tion of glutamate by glutamate dehydrogenase (GDH) was coupled to the production of formazan and formazan precipitation was used for colorim etric visualization. The method was tested and validated with gelatin model sections with known glutamate concentrations. Calibration graphs showed linear relationships with high correlation coefficients (>96%) between glutamate concentrations or section thickness and absorbance values. The method was reproducible, with a constant percentage of 60 +/- 5% of glutamate being converted in gelatin model sections containi ng glutamate concentrations of 2 mM and higher. Glutamate concentratio ns were estimated in periportal, intermediate, and pericentral zones o f liver lobules that contain low, intermediate, and high GDH activity, respectively. In fed adult mate rat livers, periportal zones containe d the highest concentrations of glutamate (similar to 14 mM) and inter mediate and pericentral zones similar to 13 and 9 mM, respectively. On starvation. glutamate concentrations increased only in the small rim of pericentral cells that express glutamine synthetase, to similar to 15 mM. In livers of fetal and newborn rats, glutamate was homogeneousl y distributed, with a concentration of similar to 5 mM. In suckling ra t liver, distribution of glutamate was still homogeneous but the conce ntration was increased to similar to 8 mM. These glutamate distributio n patterns were in agreement with those detected immunohistochemically .