LETTER |
Correspondence to: Dirk Pette, Faculty of Biology, U. of Konstanz, D-78457 Konstanz, Germany.
Image analysis is increasingly used for quantitative evaluation of histochemically assessed enzyme activity. Two methods are presently used: (a) estimation of enzyme activity by image analysis of tissue sections stained for a specific enzyme reaction (single-point or endpoint measurement), (b) determination of maximal initial reaction rates by monitoring changes in optical density during the early phase of the reaction (kinetic microphotometric measurement).
Determination of enzyme activity by single time point measurement is feasible if the reaction follows zero-order kinetics. However, this condition may be fulfilled only during the initial phase of the reaction and may vary for the same enzyme according to its cellular or tissue distribution. This phenomenon is illustrated in Figure 1, which compares microphotometrically monitored reaction rates of succinate dehydrogenase (SDH) in muscle fibers with high, intermediate, and low activity. Obviously, reaction rates are linear with time only during the first 12 min and then level off. After 12 min they have declined to approximately 48% (high), 56% (intermediate), and 61% (low) of the initial rates. Because nonspecific formazan production was monitored individually for each fiber in a parallel section and subtracted from the reaction rate in the presence of substrate, decreasing reaction rates could not have resulted from "nothing dehydrogenase" artifacts. Similar results were obtained when corrections for nothing dehydrogenase were performed for each fiber by measurements on the same section using assay mixtures without and with succinate (
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The present results and previous findings from our laboratory (
In summary, evaluations of endpoint measurements may yield reliable results only when incubation periods are restricted to that phase of the reaction during which enzyme activity and product formation display a linear relationship. This condition must be individually validated for each measurement. This is easily ascertained by kinetic microphotometry. Because image analysis greatly facilitates such measurements, we see no reason not to apply this method to studies in quantitative enzyme histochemistry.
Acknowledgments
Supported by Deutsche Forschungsgemeinschaft Pe 62/18-2 and Pe 62/25-1.
Received for publication August 18, 1997; accepted October 23, 1997.
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