A novel quantitative histochemical assay to measure endogenous substrate concentrations in tissue sections....

A quantitative histochemical assay has been developed for measurement of endogenous substrate concentrations in cryostat sections using a colorimetric visualization technique. Model sections of frozen gelatin solutions with known concentrations of glucose-6-phosphate (G6P) were sandwiched with a second cryostat section containing glucose-6-phosphate dehydrogenase (G6PDH) and all other compounds (with the exception of G6P) that are necessary for the demonstration of G6PDH activity with a tetrazolium salt method. After 60 min of incubation, G6P was converted with concomitant formazan production. The amount of formazan generated was measured cytophotometrically and used as a parameter of the G6P concentration in the first section. A calibration graph was obtained with a high correlation coefficient, allowing the conversion of mean integrated absorbance values into absolute substrate concentrations. The method was highly reproducible, and the recovery of G6P was 85 +/- 4% irrespective section thickness (4-20 microns) and G6P concentration (0.08-1.6 mM) in the sections. The sensitivity of the tetrazolium-linked method appeared to be 100 microM in 20 microns thick sections. This sensitivity enables the measurement of physiological substrate concentrations in tissue sections. Spatial resolution was approximately 150 microns, indicating a relatively high rate of diffusion of G6P during the reaction. The model study shows that the method described here allows the quantitative determination of substrate concentrations in tissue sections. These endogenous substrate concentrations are necessary for the calculation of local metabolic fluxes when determined in combination with local enzyme activities and kinetics, thus giving a more accurate reflection of in situ metabolic heterogeneity of tissues.