Fractionated illumination in oesophageal ALA-PDT: effect on ferrochelatase activity

Abstract

Background and objective: Administration of 5-aminolevulinic acid (ALA) induces accumulation of the photosensitive compound protoporphyrin IX (PpIX) in certain tissues. PpIX can be used as photosensitizer in photodynamic therapy (PDT). More selective or higher PpIX accumulation in the area to be treated could optimize the results of ALA-PDT. Porphobilinogen deaminase (PBGD) is rate-limiting in PpIX formation whereas ferrochelatase converts PpIX into haem by chelation of ferrous iron into PpIX. This results in a moment of close interaction (ferrochelatase binding to PpIX) during which ferrochelatase could selectively be destroyed resulting in an increased PpIX concentration. The aim of the present study was to investigate whether illumination before PDT can selectively destroy ferrochelatase, and whether this results in higher PpIX accumulation and thereby increases the PDT effect. Furthermore, the effect of a second ALA dose was tested. Study design/materials and methods: Oesophageal tissue of 60 rats were allocated to 2 groups of 30 animals each. In one group, enzyme and PpIX measurements were performed after ALA administration (200 mg/kg orally, n=20), or a second dose of 200 mg/kg ALA at 4 h ( n=10), half of each group with and without illumination at 1 h with 12.5 J/cm diffuser length. In the second group, PDT was performed. Ten animals were illuminated at 3 h after ALA administration with 20 ( n=5) or 32.5 J/cm ( n=5), 10 animals were illuminated at 1 h (12.5 J/cm) and received intra-oesophageal PDT treatment (20 J/cm) at 3 h ( n=5) or 4 h ( n=5) after ALA. Additionally, 10 animals received a second dose of 200 mg/kg ALA at 4 h and were illuminated (20 J/cm) at 7 h after the first dose of ALA with ( n=5) or without ( n=5) illumination at 4 h (12.5 J/cm). Results: Illumination with 12.5 J/cm at 1 h after ALA administration caused inhibition of the activity of ferrochelatase at 3 and 4 h after ALA ( P=0.02 and P<0.001, respectively), but not at 7 h ( P=0.3). In animals sacrificed at 4 h the ratio PBGD:ferrochelatase was higher in animals illuminated at 1 h compared to non-illuminated animals ( P<0.001). PpIX concentration was highest (42.7±3.2 pmol/mg protein) at 3 h after ALA administration and did not increase by illumination at 1 h. Administration of a second dose of ALA did not result in higher PpIX accumulation. After PDT, no difference in epithelial or muscular damage was found between the various groups. Conclusion: Illumination at 1 h after ALA administration can cause selective destruction of ferrochelatase, resulting in a higher ratio of PBGD:ferrochelatase. This does not result in accumulation of more porphyrins, even when a second dose of ALA is given. Therefore, under the conditions used in this study fractionated illumination does not enhance ALA-PDT-induced epithelial ablation of the rat oesophagus.