Introduction
Cushing’s Disease (CD) is caused by millimeter-sized corticotropinomas (microadenomas) that lead to supraphysiological levels of glucocorticoid. Up to 40% of microadenomas are not visualized on gold-standard MR imaging. Pituitary adenomas metabolize exogenous 5-ALA (an endogenous metabolite) to protoporphyrin IX (PpIX) at rates 20-50 times higher compared with normal tissues. PpIX intensely fluoresces red (635nm) when excited with blue light (375-440nm), enabling its use as an intraoperative fluorescence imaging agent. 5-ALA is now an FDA approved prodrug. We examined the efficacy of ALA-induced-PpIX fluorescence in human derived adenomatous and normal pituitary samples. We explored the modulation of PpIX conversion with CRH or dexamethasone (DEX), and subcellular localization of PpIX.
Methods
We used flow cytometry for PpIX intensity analysis. A human-derived corticotropinoma, it’s adjacent normal gland, murine normal pituitary cells, and AtT20 cells were incubated with 5-ALA (300 nM) with/without DEX (1µM) or CRH (50nM). For confocal microscopy, live cells imaged for PpIX (405nm/615nm) and mitochondrial (550nm/615nm) fluorescence.
Results
We found a 10-fold-increase in 5-ALA induced PpIX fluorescence intensity in human-derived adenomatous compared to adjacent normal pituitary tissue (n=1, p\u3c0.05). AtT-20 cell lines (n=6, p\u3c0.05) fluoresced 7-fold more intensely compared to normal murine pituitary tissue (n=3, p\u3c0.05). The addition of DEX, before or after 5-ALA exposure, increased the fluorescence intensity by 31% (n=4, p\u3c0.05). The addition of CRH did not have a significant effect on 5-ALA fluorescence (n=3, p\u3c0.05). We saw localization of 5-ALA to mitochondria, and mitochondrial disruption in 5-ALA treated At-T20s.
Conclusions
Our results support the use of 5-ALA for fluorescence guided resection in hormone secreting microadenomas. The supraphysiological levels of glucocorticoids, as seen in CD, may enhance the 5-ALA fluorescence in corticotropinomas. We confirm the mitochondrial localization and disruption by 5-ALA, a basis for photodynamic therapy
