Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the experimentally injured rabbit cornea and tear fluid using the sensitive substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC)

Comparative histochemical and biochemical studies on acid beta-galactosidas e activity in the rabbit eye after various experimental injuries were perfo rmed using the same sensitive fluorogenic substrate beta-galactoside-4-trif luoromethylumbelliferyl (HFC), The aim of the study was to examine whether the severity of corneal damage corresponds with the level of the enzyme act ivity in the tear fluid. As until recently the substrate beta-galactoside-4 -HFC had not been used for the histochemical detection of acid beta-galacto sidase in the cornea, results obtained with this substrate in a fluorescent method were compared in parallel cryostat sections with results obtained u sing the substrate 5-bromo-4-chloro-3-indoxyl beta-galactoside in the indig ogenic method (previously shown to be very sensitive for the detection of a cid beta-galactosidase activity in the cornea). Both methods revealed simil ar localization and changes in enzyme activity; using beta-galactoside-4-HF C an acceptable cellular localization was achieved. For the measurement of acid beta-galactosidase activity in the tear fluid a semiquantitative bioch emical method was elaborated using filter paper punches with the substrate (beta-galactoside-4-HFC) soaked with tears and incubated at 37 degrees C. T he time of the first appearance of a greenish-yellow fluorescence (enzyme p ositivity) was recorded by UV lamp and compared with the appearance of fluo rescence in calibrated punches containing known acid beta-galactosidase act ivities. The results show that beta-galactoside-4-HFC is useful for the bio chemical assessment of acid beta-galactosidase activity in the tear fluid. Comparing histochemical and biochemical results, it can be concluded that i ncreased enzymatic activity in tears parallels the severity of corneal dama ge. Further studies are necessary to evaluate whether the detection of acid beta-galactosidase activity in tears might be useful for diagnostic purpos es in humans.