Chromatography and spectrofluorometry of brain fluorophores in neuronal ceroid lipofuscinosis (NCL).
The aim of the present work was to develop a chromatographic system for the separation of individual fluorophores extracted from neuronal ceroid lipofuscinosis (NCL) brain and isolated storage bodies. Extracts from gray matter were best resolved on silica-gel HPTLC plates using a mixture of chloroform/methanol/water (55:45:10 by vol.). Two other chromatographic systems were tested which gave poorer separation. Corrected fluorescence spectra were obtained on the original extract and fluorescence intensity, especially at longer wavelengths was increased in both samples. Yellow and blue fluorophores were detected on HPTLC plates using a primary violet and secondary yellow filter with cut-off levels of 400 and 520 nm, respectively. Plates were photographed at 20 min, 2 h and 1 week after chromatography. With this filter system, up to 12 yellow bands of differing intensity were observed at 20 min but with time, some of these changed to blue as a result of autoxidation. NCL tissues emit yellow fluorescence when viewed under light microscopy, however extracted material did not demonstrate a distinct peak in this region of the spectrum which should be around 575 nm. HPTLC confirmed this observation and time studies revealed that autoxidation changes occur and must be carefully controlled to reduce artifacts. The discrepancy between extracted and non-extracted observations may be the result of superposition of multiple fluorophores with differing maxima and/or a self-absorption phenomenon. The combination of chromatographic separation and spectral analysis as described in this study, may be a valuable technique to further clarify the characteristics of compound fluorescent lipopigments. It is suggested that NCL fluorophores of human brain differ in their properties from other models.