A Quantitative Histochemical Study of Alkaline Phosphatase Activity in Isolated Rat Duodenal Epithelial Cells

A simple separation method enabling the quantification of alkaline phosphatase activity in unfixed, isolated, individual, duodenal epithelial cells has been presented. The activity of intestinal brush border-bound alkaline phosphatase has been demonstrated using naphthol AS-BI phosphate as a substrate and hexazotized New Fuchsin as a simultaneous coupling agent. The amount of final reaction product, as measured cytophotometrically, increases linearly with incubation time (up to 10 min) and with substrate concentration (up to 0.4 mM). Maximum enzyme activity was obtained at pH 8.9. Variation of the substrate concentration revealed the kinetic parameters for naphthol AS-BI phosphate as K_m = 0.17 ± 0.015 and V_max = 13.9 ± 1.38. The specificity of the enzyme reaction was confirmed by the complete inhibition of the enzyme activity in the presence of l-cysteine (10 mm) and 80% inhibition with L - phenylalanine (30 mM). Comparison of alkaline phosphatase activity in 8-m cryostat sections (beginning at the tip and proceeding to the cryptal part) along the villus axis, with the activity of individual cells obtained by successive separation, revealed similar values of the percentage quotient derived from the entire activities in these two different methods. This suggests that the presented separation procedure gives rise to isolation of the respective cells from the corresponding areas of the villus. Finally, the isolated cells can be used as a valuable tool for the quantitative analysis of alkaline phosphatase activity along the length of the villus.