engleski

Analysis of Formalin Fixation Influence on Ganglioside Composition of Calf Brain

Ganglioside composition and expression in brain tissue change in various physiological and pathophysiological conditions. We explored to which extent the brain tissue fixation using lower concentrations of formaldehyde (FA) solutions influences the procedure of ganglioside extraction and purification from fixed tissue i.e. whether there are differences in content and composition of gangliosides isolated from fixed vs. fresh tissue. Identical aliquots of the 10% fresh calf brain tissue homogenates were fixed in 1.5%, 4% and 10% solutions of FA for five weeks, while aliquots of fresh homogenates were used as controls. Gangliosides were extracted from the testing-series of homogenates and purified. The total content of isolated gangliosides was determined by spectrophotometric assay. The composition of ganglioside mixtures was analyzed by thin layer chromatography and the proportions of individually separated fractions were determined by densitometry. Comparing to the fresh tissue, the total content of gangliosides isolated from tissue fixed in 1.5% FA solution was slightly decreased (5.4%), while the total contents of gangliosides isolated from tissues fixed in 4% and 10% FA solutions were lower for 9.8% and 14.9%, respectively. Ganglioside mixtures from fixed tissues contained all individual (chromatographically separable) fractions present in the ganglioside mixture from fresh tissue, although in the ganglioside mixture from tissue fixed in 10% FA solution only traces of GD3 and GQ1b remained. The observed changes in the composition of total gangliosides isolated from fixed vs. fresh tissue were: significant to high decrease of GD3 and GQ1b proportions and a moderate decrease of O-Ac-GT1b proportion ; additionally, a moderate decrease of GT1b proportion was detected only in the ganglioside sample from tissue fixed in 10% FA solution. The results show that the fixation of brain tissue in 1.5% FA solution does not significantly affect the procedure of ganglioside isolation and does not cause significant changes in the quantity and composition of isolated gangliosides, except reduced GD3 abundance. This investigation provides answers what are the restrictions for utilization of fixed tissue samples as biological material for comparative analysis of the content and composition of gangliosides, if fresh material is not available.

gangliosides ; calf brain ; formalin fixation

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