In Situ Measurement of Glutamate Concentrations in the Periportal, Intermediate, and Pericentral Zones of Rat Liver

Abstract

We developed a quantitative histochemical assay for measurement of local glutamate concentrations in cryostat sections of rat liver. Deamination of glutamate by glutamate dehydrogenase (GDH) was coupled to the production of formazan and formazan precipitation was used for colorimetric 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 containing glutamate concentrations of 2 mM and higher. Glutamate concentrations were estimated in periportal, intermediate, and pericentral zones of liver lobules that contain low, intermediate, and high GDH activity, respectively. In fed adult male rat livers, periportal zones contained the highest concentrations of glutamate (∼14 mM) and intermediate and pericentral zones ∼13 and 9 mM, respectively. On starvation, glutamate concentrations increased only in the small rim of pericentral cells that express glutamine synthetae, to ∼15 mM. In livers of fetal and newborn rats, glutamate was homogeneously distributed, with a concentration of ∼5 mM. In suckling rat liver, distribution of glutamate was still homogeneous but the concentration was increased to ∼8 mM. These glutamate distribution patterns were in agreement with those detected immunohistochemically.