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

Abstract

Comparative histochemical and biochemical studies on acid β-galactosidase activity in the rabbit eye after various experimental injuries were performed using the same sensitive fluorogenic substrate β-galactoside-4- trifluoromethylumbelliferyl (HFC). The aim of the study was to examine whether the severity of corneal damage corresponds with the level of the enzyme activity in the tear fluid. As until recently the substrate β-galactoside-4- HFC had not been used for the histochemical detection of acid β-galactosidase in the cornea, results obtained with this substrate in a fluorescent method were compared in parallel cryostat sections with results obtained using the substrate 5-bromo-4-chloro-3-indoxyl β-galactoside in the indigogenic method (previously shown to be very sensitive for the detection of acid β- galactosidase activity in the cornea). Both methods revealed similar localization and changes in enzyme activity; using β-galactoside-4-HFC an acceptable cellular localization was achieved. For the measurement of acid β-galactosidase activity in the tear fluid a semiquantitative biochemical method was elaborated using filter paper punches with the substrate (β-galactoside- 4-HFC) soaked with tears and incubated at 37 "C. The time of the first appearance of a greenishyellow fluorescence (enzyme positivity) was recorded by UV lamp and compared with the appearance of fluorescence in calibrated punches containing known acid β-galactosidase activities. The results show that β-galactoside- 4-HFC is useful for the biochemical assessment of acid β-galactosidase activity in the tear fluid. Comparing histochemical and biochemical results, it can be concluded that increased enzymatic activity in tears parallels the severity of corneal damage. Further studies are necessary to evaluate whether the detection of acid β-galactosidase activity in tears might be useful for diagnostic purposes in humans.