Porphyrin bleaching and PDT-induced spectral changes are irradiance dependent in ALA-sensitized normal rat skin in vivo

Photobleaching kinetics of aminolevulinic acid-induced protoporphyrin IX (P pIX) were measured in the normal skin of rats in vivo using a technique in which fluorescence spectra were corrected for the effects of tissue optical properties in the emission spectral window through division by reflectance spectra acquired in the same geometry and wavelength interval and for chan ges in excitation wavelength optical properties using diffuse reflectance m easured at the excitation wavelength, Loss of PpIX fluorescence was monitor ed during photodynamic therapy (PDT) performed using 514 nm irradiation. Bl eaching in response to irradiances of 1, 5 and 100 mW cm(-2) was evaluated, The results demonstrate an irradiance dependence to the rate of photobleac hing vs Irradiation fluence, with the lowest irradiance leading to the most efficient loss of fluorescence, The kinetics for the accumulation of the p rimary fluorescent photoproduct of PpIX also exhibit an irradiance dependen ce, with greater peak accumulation at higher irradiance. These findings are consistent with a predominantly oxygen-dependent photobleaching reaction m echanism in vivo, and they provide spectroscopic evidence that PDT delivere d at low irradiance deposits greater photodynamic dose for a given irradiat ion fluence, We also observed an irradiance dependence to the appearance of a fluorescence emission peak near 620 nm, consistent with accumulation of uroporphyrin/coproporphyrin in response to mitochondrial damage.