CELLULAR FLUORESCENCE OF THE ENDOGENOUS PHOTOSENSITIZER PROTOPORPHYRIN-IX FOLLOWING EXPOSURE TO 5-AMINOLEVULINIC ACID

Supplying 5-aminolevulinic acid (ALA), a precursor in the biosynthetic pathway to heme from an external source leads to an accumulation of t he endogenous fluorescent photosensitizer protoporphyrin IX (PPIX). Fo llowing instillation of ALA in the urinary bladder neoplastic tissue c an be discerned by fluorescence cystoscopy or treated by illumination with light of an appropriate wavelength. In order to provide a biologi cal rationale for the clinical findings, we have analyzed the capacity of three different cell lines to accumulate PPM by flow cytometry. Th ree different urothelial cell Lines, normal fibroblasts and endothelia l cells were exposed to ALA under varying conditions. Urothelial cell lines J82 and RT4, derived from malignancies of the bladder displayed fluorescence intensities 9- and 16-fold, respectively, above the fluor escence level of the normal urothelial cell line HCV29. Human umbilica l cord endothelial cells fluoresced moderately while the fibroblast ce ll line N1 exhibited a fluorescence level comparable to those of the c ancer cells. Fluoresence increased with increasing cell density and wa s also dependent on the growth of cells as monolayers or multicellular spheroids. Increasing ALA concentrations led to saturation of fluores cence after 4 h of incubation at cell type-specific fluorescence level s obtained at different ALA concentrations. Continuous incubation in m edium containing serum resulted in a linear rise of fluorescence durin g the first 4 h, which was followed by a saturation period (8-24 h) an d a renewed rise. In the case of serum depletion, fluorescence intensi ties were significantly higher and increased linearly during the entir e 48 h incubation period. By replacing serum with albumin, it could be shown that the emission of PPM into the medium in the presence of ser um is mainly caused by this protein. The ALA-induced fluorescence was predominantly perinuclear after 4 h of incubation and relocated toward the cell membrane after prolonged incubation. This study demonstrated the complexity of factors influencing the ALA-induced fluorescence an d should stimulate further research in this field.