A QUANTITATIVE HISTOCHEMICAL-STUDY OF ALKALINE-PHOSPHATASE ACTIVITY IN ISOLATED RAT DUODENAL EPITHELIAL-CELLS

A simple separation method enabling the quantification of alkaline pho sphatase activity in unfixed, isolated, individual, duodenal epithelia l cells has been presented. The activity of intestinal brush border-bo und alkaline phosphatase has been demonstrated using naphthol AS-BI ph osphate as a substrate and hexazotized New Fuchsin as a simultaneous c oupling agent. The amount of final reaction product, as measured cytop hotometrically, increases linearly with incubation time (up to 10 min) and with substrate concentration (up to 0.4 mM). Maximum enzyme activ ity was obtained at pH 8.9. Variation of the substrate concentration r evealed 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 activ ity in the presence of L-cysteine (10 mm) and 80% inhibition with L-ph enylalanine (30 mM). Comparison of alkaline phosphatase activity in 8- mu m cryostat sections (beginning at the tip and proceeding to the cry ptal part) along the villus axis, with the activity of individual cell s obtained by successive separation, revealed similar values of the pe rcentage quotient derived from the entire activities in these two diff erent methods. This suggests that the presented separation procedure g ives rise to isolation of the respective cells from the corresponding areas of the villus. Finally, the isolated cells can be used as a valu able tool for the quantitative analysis of alkaline phosphatase activi ty along the length of the villus. (C) 1998 Chapman & Hall.