Spectrofluorometric Determination of Protoporphyrin IX in Cells Using Acridine as Internal Standard

Photodynamic therapy (PDT) has been used for diagnosis and treatment of patients with various malignant tumors, including cancers of the skin, gastrointestinal tract, lung and uterus. Recently, PDT with using 5-aminolevulinic acid (ALA) for cutaneous diseases has been attracted considerable attentions, because skin is easily accessible to light and drugs. Since ALA was used as a pre-drug of the photosensitizer in PDT research by Kenedy et al. in 1990, a number of ALA ester derivatives have been synthesized and applied to PDT. ALA and its ester derivatives are precursors of protoporphyrin IX (PpIX) in the biosynthetic pathway of heme. They are normally biosynthesized in vivo from glycine and succinyl-coenzyme A in mitochondria and then converted into the actual photosensitizer, PpIX. PpIX produced through the heme biosynthetic pathway emits fluorescence strongly and photobleaches rapidly under production of singlet oxygen. The amount of ALA-induced porphyrins can be generally determined by measuring the fluorescence under various experimental conditions. Most papers have reported that the amount of fluorescence was determined by the measurement of fluorescence intensity at the maximum emission wavelength. Fluorescence spectra were measured by single-beam operation in which a sample is examined to determine the amount of light emitted at a given wavelength. Unstable light source and uneven intensities can cause the false peaks in the spectrum. Equally serious is the variation of the sensitivity of the photomultiplier tubes with regard to wavelength. Moreover, the analytical method for the determination of the fluorescence intensity at a particular wavelength is less sensitive than that of fluorescence spectrum area. The aim of this study is to present a highly sensitive and convenient spectrofluorometric method using an internal standard for the determination of the amount of PpIX produced by ALA in the various tumor and normal cells.