Clearance mechanism of protoporphyrin IX from mouse skin after application of 5-aminolevulinic acid
Introduction
5-Aminolevulinic acid (ALA) or its esters induced protoporphyrin IX (PpIX) synthesis in neoplastic cells is used for fluorescence diagnostics, fluorescence-guided surgery and photodynamic therapy (PDT) [1], [2], [3], [4], [5]. Under normal conditions the levels of PpIX are tightly regulated, mainly by haem control of the ALA synthase activity, which is the first and also the rate-limiting enzyme of the haem synthetic pathway [6]. When applied topically or systemically, ALA bypasses the negative feedback control and induces intracellular accumulation of PpIX [1]. Since the other enzymes are essentially non-limiting, and not feedback regulated, exogenous ALA metabolism leads to overproduction of PpIX. PDT efficiency depends on intracellular PpIX levels. These levels depend on the balance between PpIX synthesis and PpIX clearance by the action of ferrochelatase in the presence of iron into haem, or by PpIX efflux from cells to blood stream. The synthesis of PpIX in vitro and in vivo has been systematically investigated in order to find methods to increase the accumulation of PpIX in target cells. Extracellular and intracellular ALA concentrations, cell type (number of mitochondria, haem synthesis enzymes activity), physiological factors (temperature, pH), and iron availability regulate the synthesis of PpIX [7]. Endogenously produced PpIX is cleared from the tissues within 24–48 h after topical and systemic application [8], [9], [10]. Due to the fast clearance of PpIX only few studies have investigated its intracellular clearance. The clearance rates of PpIX vary widely between patients with the same or different disorders [8], [11]. A deeper understanding of PpIX clearance mechanisms in the early stages after ALA application may indicate how we can reduce PpIX clearance rates and improve PDT outcome.
After ALA administration in vitro and in vivo PpIX is synthesized in the mitochondria [1]. Then PpIX diffuses from the mitochondria and, being lipophilic, is relocalized in cell membranes and cytoplasm [12], [13], [14]. In vitro, extracellular serum proteins bind PpIX molecules and speed up leakage from the cells (PpIX efflux) [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]. In the presence of serum both PpIX clearance mechanisms, cellular efflux and conversion into haem, take place in vitro. In the absence of serum, PpIX is mostly converted into haem [25], [26]. So far, little emphasis has been focused on the detailed clearance mechanisms of PpIX after ALA application and its control mechanisms in vivo.
Ferrochelatase converts PpIX into haem by inserting ferrous iron into PpIX, and the presence of iron chelators alone promotes the accumulation of PpIX [27]. The comparison of clearance rates of iron chelator-induced PpIX and ALA-induced PpIX may help to understand the clearance mechanisms of PpIX. If PpIX is mostly converted into haem (not via the efflux of PpIX from cells into blood), iron chelator-induced PpIX will be converted into haem much slower than ALA-induced PpIX due to the iron deficiency. Topical application of cream containing a well known iron chelator, ethylenediaminetetraacetic acid (EDTA), has been chosen to induce PpIX and study its clearance.
In the present work we report that PpIX induced by exogenous application of ALA in skin is converted into haem rather than diffuse from the skin into blood stream.
Section snippets
Preparation of the drugs
The used drugs 5-aminolevulinic acid (ALA) hydrochloride (Sigma Chemical Co., St. Louis, MO) or ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co., St. Louis, MO) were directly dissolved phosphate buffered saline (PBS) or in a base cream (Unguentum M, Merck, Darmstadt, Germany). Around 50 μl of 200 mg/kg ALA was injected intraperitoneally (i.p.) into mice. Approximately 100 mg of 20% (w/w) ALA or 20% EDTA cream was applied topically on a single spot (area around 1 cm2) on the right flank of
PpIX induction by ALA or EDTA
Normalized fluorescence emission spectra, registered from normal mouse skin after topical or systemic application of ALA and EDTA, are shown in Fig. 1. These spectra are very similar in shape and both have their maxima at 636 nm and 705 nm, confirming that PpIX is the main fluorescent porphyrin induced by EDTA and ALA.
The maximum fluorescence intensities of PpIX after systemic and topical application of ALA were reached at around 3 h and 6–24 h, respectively (Fig. 2, Fig. 3). PpIX fluorescence
Discussion
PpIX is a highly hydrophobic molecule, and one can expect that it is mainly bound or retained in lipophilic structures in cells and tissues. Supposedly, it is not cleared from the body by renal excretion. However, ALA-induced PpIX is cleared from the body within 24–48 h [8], [9], [10]. Thus, a question of the basic pharmacokinetics, especially that of the “clearance” phase of PpIX is of large scientific interest. A number of investigators have already paid attention to the conversion of PpIX
Conclusions
ALA application mode (topical versus systemic) and skin viability (dead versus alive) have no influence on PpIX decay in normal mouse skin. Since ferrochelatase catalyzes the insertion of iron ions into the PpIX molecule to produce haem, the labile intracellular iron pool influences the PpIX levels. In our study EDTA was used to diminish the availability of intracellular iron pool. This delayed the conversion of PpIX into haem. The incorporation of iron into PpIX also depends on skin
Acknowledgements
The present work was supported by the South-Eastern Norway Regional Health Authority and the Norwegian Cancer Society and Oslo University Hospital.
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