Cellular Fluorescence of the Endogenous Photosensitizer Protoporphyrin Ix Following Exposure to 5‐aminolevulinic Acid

Abstract

Abstract— Supplying 5‐aminolevulinic acid (ALA), a precursor in the biosynthetic pathway to heme from an external source leads to an accumulation of the endogenous fluorescent photosensitizer protoporphyrin IX (PPIX). Following instillation of ALA in the urinary bladder neoplastic tissue can be discerned by fluorescence cystoscopy or treated by illumination with light of an appropriate wavelength. In order to provide a biological rationale for the clinical findings, we have analyzed the capacity of three different cell lines to accumulate PPIX by flow cytometry. Three different urothelial cell lines, normal fibroblasts and endothelial 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 fluorescence level of the normal urothelial cell line HCV29. Human umbilical cord endothelial cells fluoresced moderately while the fibroblast cell line Nl exhibited a fluorescence level comparable to those of the cancer cells. Fluoresence increased with increasing cell density and was also dependent on the growth of cells as monolayers or multicellular spheroids. Increasing ALA concentrations led to saturation of fluorescence after 4 h of incubation at cell type‐specific fluorescence levels obtained at different ALA concentrations. Continuous incubation in medium containing serum resulted in a linear rise of fluorescence during the first 4 h, which was followed by a saturation period (8–24 h) and a renewed rise. In the case of serum depletion, fluorescence intensities were significantly higher and increased linearly during the entire 48 h incubation period. By replacing serum with albumin, it could be shown that the emission of PPIX into the medium in the presence of serum 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 and should stimulate further research in this field.

Regulation of 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation in Human Urothelial Carcinomas

“…Especially in the urinary tract, the quantitative analysis of fluorescence spectra showed that PpIX was enhanced 17-fold in urothelial neoplasia compared with normal mucosa [4,36,37] . Thus, the physicochemical properties of PpIX have been used for PDD and PDT of patients with tumors [38] .…”

The Quality of 5-Aminolevulinic Acid-Induced Photodynamic Diagnosis and Transurethral Resection of Bladder Tumors: Does the Urologist Play a Role?

“…Nonmalignant bladder lesions can produce fluorescence but, to our knowledge, the relative fluorescence intensity of false-positive biopsies has not yet been investigated. On the other hand, major variations in PPIX accumulation, with a 10- to 50-fold difference, can be observed in various cell lines [23,24] which is attributed to a difference in metabolic activity [25]. …”

Identification of Carbonic Anhydrase IX as a Novel Target for Endoscopic Molecular Imaging of Human Bladder Cancer

“…Hexaminolevulinate is approved for single administration on account of potential drug hypersensitivity with repeat exposure. Other technical limitations include a decreased optical management with incomplete hemostasis and a photobleaching effect causing loss of fluorescence [27]. Undeniably, wide-field fluorescence imaging of CAIX offers potential advantages over the currently approved blue-light cystoscopy with protoporphyrin.…”