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• W1984225067 abstract "Topical 5-aminolevulinic acid is used for the fluorescence-based diagnosis and photodynamic treatment of superficial precancerous and cancerous lesions of the skin. Thus, we investigated the kinetics of 5-aminolevulinic acid-induced fluorescence and the mechanisms responsible for the selective formation of porphyrins in tumors in vivo. Using amelanotic melanomas (A-Mel-3) grown in dorsal skinfold chambers of Syrian golden hamsters fluorescence kinetics were measured up to 24 h after topical application of 5-aminolevulinic acid (1%, 3%, or 10%) for 1 h, 4 h, or 8 h by intravital microscopy (n = 54). Maximal fluorescence intensity in tumors after 1 h application (3% 5-aminolevulinic acid) occurred 150 min and after 4 h application (3% 5-aminolevulinic acid) directly thereafter. Increasing either concentration of 5-aminolevulinic acid or application time did not yield a higher fluorescence intensity. The selectivity of the fluorescence in tumors decreased with increasing application time. Fluorescence spectra indicated the formation of protoporphyrin IX (3% 5-aminolevulinic acid, 4 h; n = 3). The simultaneous application of 5-aminolevulinic acid (3%, 4 h) and glycine (20 μM or 200 μM; n = 10) reduced fluorescence in tumor and surrounding host tissue significantly. In contrast, neither decreasing iron concentration by desferrioxamine (1% and 3%; n = 10) nor inducing tetrapyrrole accumulation using 1,10-phenanthroline (7.5 mM; n = 5) increased fluorescence in tumors. The saturation and faster increase of fluorescence in the tumor together with a reduction of fluorescence by the application of glycine suggests an active and higher intracellular uptake of 5-aminolevulinic acid in tumor as compared with the surrounding tissue. Shorter application (1 h) yields a better contrast between tumor and surrounding tissue for fluorescence diagnosis. The additional topical application of modifiers of the heme biosynthesis, desferrioxamine or 1,10-phenanthroline, however, is unlikely to enhance the efficacy of topical 5-aminolevulinic acid–photodynamic therapy at least in our model. Topical 5-aminolevulinic acid is used for the fluorescence-based diagnosis and photodynamic treatment of superficial precancerous and cancerous lesions of the skin. Thus, we investigated the kinetics of 5-aminolevulinic acid-induced fluorescence and the mechanisms responsible for the selective formation of porphyrins in tumors in vivo. Using amelanotic melanomas (A-Mel-3) grown in dorsal skinfold chambers of Syrian golden hamsters fluorescence kinetics were measured up to 24 h after topical application of 5-aminolevulinic acid (1%, 3%, or 10%) for 1 h, 4 h, or 8 h by intravital microscopy (n = 54). Maximal fluorescence intensity in tumors after 1 h application (3% 5-aminolevulinic acid) occurred 150 min and after 4 h application (3% 5-aminolevulinic acid) directly thereafter. Increasing either concentration of 5-aminolevulinic acid or application time did not yield a higher fluorescence intensity. The selectivity of the fluorescence in tumors decreased with increasing application time. Fluorescence spectra indicated the formation of protoporphyrin IX (3% 5-aminolevulinic acid, 4 h; n = 3). The simultaneous application of 5-aminolevulinic acid (3%, 4 h) and glycine (20 μM or 200 μM; n = 10) reduced fluorescence in tumor and surrounding host tissue significantly. In contrast, neither decreasing iron concentration by desferrioxamine (1% and 3%; n = 10) nor inducing tetrapyrrole accumulation using 1,10-phenanthroline (7.5 mM; n = 5) increased fluorescence in tumors. The saturation and faster increase of fluorescence in the tumor together with a reduction of fluorescence by the application of glycine suggests an active and higher intracellular uptake of 5-aminolevulinic acid in tumor as compared with the surrounding tissue. Shorter application (1 h) yields a better contrast between tumor and surrounding tissue for fluorescence diagnosis. The additional topical application of modifiers of the heme biosynthesis, desferrioxamine or 1,10-phenanthroline, however, is unlikely to enhance the efficacy of topical 5-aminolevulinic acid–photodynamic therapy at least in our model. 5-aminolevulinic acid protoporphyrin IX 5-aminolevulinic acid (ALA), a precursor of heme, has stimulated enormous interest as endogenous photosensitizer for fluorescence diagnosis and photodynamic therapy of tumors after topical application. Bypassing the feedback control mechanism of the heme biosynthesis, the administration of ALA induces the rather selective formation of porphyrins in epithelial tissue (Kennedy et al., 1990Kennedy J.C. Pottier R.H. Pross D.C. Photodynamic therapy with endogenous protoporphyrin IX. basic principles and present clinical experience.J Photochem Photobiol B Biol. 1990; 6: 143-148Crossref PubMed Scopus (1339) Google Scholar;Fritsch et al., 1998Fritsch C. Goerz G. Ruzicka T. Photodynamic therapy in dermatology.Arch-Dermatol. 1998; 134: 207-214Crossref PubMed Scopus (261) Google Scholar). The hydrophilic molecule ALA can be administered either systemically (Grant et al., 1993Grant W.E. Hopper C. MacRobert A.J. Speight P.M. Bown S.G. Photodynamic therapy of oral cancer: photosensitation with systemic aminolaevulinic acid.Lancet. 1993; 342: 147-148Abstract PubMed Scopus (240) Google Scholar;Regula et al., 1995Regula J. MacRobert A.J. Gorchein A. et al.Photosensitisation and photodynamic therapy of oesophageal, duodenal and colorectal tumors using 5-aminolaevulinic acid induced protoporphyrin IX-a pilot study.Gut. 1995; 36: 67-75Crossref PubMed Scopus (307) Google Scholar;Fritsch et al., 1997Fritsch C. Abels C. Goetz A.E. et al.Porphyrins preferentially accumulate in a melanoma following intravenous injection of 5-aminolaevulinic acid.Biol Chem. 1997; 378: 51-57Crossref PubMed Scopus (30) Google Scholar) or topically (Szeimies et al., 1994Szeimies R.M. Sassy T. Landthaler M. Penetration potency of topical applied δ-aminolevulinic acid for photodynamic therapy of basal cell carcinoma.Photochem Photobiol. 1994; 59: 73-76Crossref PubMed Scopus (135) Google Scholar;Karrer et al., 1995Karrer S. Szeimies R.M. Hohenleutner U. Heine A. Landthaler M. Unilateral localized basaliomatosis: treatment with topical photodynamic therapy after application of 5-aminolevulinic acid.Dermatology. 1995; 190: 218-222Crossref PubMed Scopus (36) Google Scholar;Fink-Puches et al., 1997Fink-Puches R. Hofer A. Smolle J. Kerl H. Wolf P. Primary clinical response and long-term follow-up of solar keratoses treated with topically applied 5-aminolevulinic acid and irradiation by different wave bands of light.J Photochem Photobiol B. 1997; 41: 145-151Crossref PubMed Scopus (91) Google Scholar). To avoid potential side-effects of the systemic administration of ALA, topically applied ALA is preferred and is already used clinically for the detection of occult neoplasms by means of the induced fluorescence (Kriegmair et al., 1994Kriegmair M. Baumgartner R. Knüchel R. et al.Photodynamische Diagnose urothelialer Neoplasien nach intravesikaler Instillation von 5-Aminolävulinsäure.Urologie. 1994; 33: 270-275PubMed Google Scholar;Leunig et al., 1996Leunig A. Rick K. Stepp H. Goetz A.E. Baumgartner R. Feyh J. Fluorescence photodetection of lesions in the oral cavity following topical application of 5-aminolaevulinic acid.Laryngol Rhinol Otol. 1996; 75: 459-464Crossref Scopus (32) Google Scholar) and for the treatment of different malignancies by means of the induced porphyrins (Kennedy and Pottier, 1992Kennedy J.C. Pottier R.H. Endogenous protoporphyrin IX, a clinical useful photosensitizer for photodynamic therapy.J Photochem Photobiol B Biol. 1992; 14: 275-292Crossref PubMed Scopus (1073) Google Scholar;Grant et al., 1993Grant W.E. Hopper C. MacRobert A.J. Speight P.M. Bown S.G. Photodynamic therapy of oral cancer: photosensitation with systemic aminolaevulinic acid.Lancet. 1993; 342: 147-148Abstract PubMed Scopus (240) Google Scholar;Wolf et al., 1993Wolf P. Rieger E. Kerl H. Topical photodynamic therapy with endogenous porphyrins after application of 5-aminolaevulinic acid.J Am Acad Dermatol. 1993; 28: 17-21Abstract Full Text PDF PubMed Scopus (340) Google Scholar;Lui et al., 1995Lui H. Salasche S. Kollias N. Wimberly J. Flotte T.M.C. Lean D. Anderson R.R. Photodynamic therapy of nonmelanoma skin cancer with topical aminolaevulinic acid: a clinical and histologic study.Arch Dermatol. 1995; 131: 737-738Crossref PubMed Scopus (66) Google Scholar;Regula et al., 1995Regula J. MacRobert A.J. Gorchein A. et al.Photosensitisation and photodynamic therapy of oesophageal, duodenal and colorectal tumors using 5-aminolaevulinic acid induced protoporphyrin IX-a pilot study.Gut. 1995; 36: 67-75Crossref PubMed Scopus (307) Google Scholar;Fromm et al., 1996Fromm D. Kessel D. Webber J. Feasibility of photodynamic therapy using endogenous photosensitization for colon cancer.Arch Surg. 1996; 131: 667-669Crossref PubMed Scopus (38) Google Scholar;Fink-Puches et al., 1997Fink-Puches R. Hofer A. Smolle J. Kerl H. Wolf P. Primary clinical response and long-term follow-up of solar keratoses treated with topically applied 5-aminolevulinic acid and irradiation by different wave bands of light.J Photochem Photobiol B. 1997; 41: 145-151Crossref PubMed Scopus (91) Google Scholar,Fink-Puches et al., 1998Fink-Puches R. Soyer H.P. Hofer A. Kerl H. Wolf P. Long-term follow-up and histological changes of superficial nonmelanoma skin cancers treated with topical delta-aminolevulinic acid photodynamic therapy.Arch Dermatol. 1998; 134: 821-826Crossref PubMed Scopus (142) Google Scholar;Gossner et al., 1998Gossner L. Stolte M. Sroka R. Rick K. May A. Hahn E.G. Ell G. Photodynamic ablation of high grade dysplasia and early cancer in Barrett’s esophagus by means of 5-aminolevulinic acid.Gastroenterology. 1998; 114: 448-455Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). The major advantage of the so-called endogenous photosensitizer ALA is the unique selectivity of the induced porphyrins not achieved with other sensitizers up to now. Whether the selectivity is due to an increased intracellular uptake, an increased porphyrin synthesis or a reduced activity of the enzyme ferrochelatase is not clear. Using the amelanotic melanoma of the hamster growing in a transparent dorsal skinfold chamber the pharmacokinetics of ALA-induced fluorescence in vivo following systemic administration of ALA were determined and revealed that the tumor selectivity is not due to a reduced activity of the enzyme ferrochelatase (Fritsch et al., 1998Fritsch C. Goerz G. Ruzicka T. Photodynamic therapy in dermatology.Arch-Dermatol. 1998; 134: 207-214Crossref PubMed Scopus (261) Google Scholar;Abels et al., 1994Abels C. Heil P. Dellian M. Kuhnle G.E.H. Baumgartner R. Goetz A.E. In vivo kinetics and spectra of 5-aminolaevulinic acid induced fluorescence in an amelanotic melanoma of the hamster.Br J Cancer. 1994; 70: 826-833Crossref PubMed Scopus (77) Google Scholar). To elucidate the mechanism of selectivity of the ALA-induced porphyrins and perhaps to increase the selectivity we investigated the effects of the amino acid glycine (Richards and Scott, 1961Richards F.F. Scott J.J. Glycine metabolism in acute porphyria.Clin Sci. 1961; 20: 387-400PubMed Google Scholar) as well as of the iron chelator desferrioxamine (Fijan et al., 1995Fijan S. Hönigsmann H. Ortel B. Photodynamic therapy of epithelial skin tumours using delta-aminolaevulinic acid and desferrioxamine.Br J Dermatol. 1995; 133: 282-288Crossref PubMed Scopus (207) Google Scholar;Berg et al., 1996Berg K. Anholt H. Bech O. Moan J. The influence of iron chelators on the accumulation of protoporphyrin IX in 5-aminolaevulinic acid-treated cells.Br J Cancer. 1996; 74: 688-697Crossref PubMed Scopus (121) Google Scholar) and of the inducer of tetrapyrroles 1,10-phenanthroline (Rebeiz et al., 1996Rebeiz N. Arkins S. Rebeiz C.A. Simon J. Zachary J.F. Kelley K.W. Induction of tumor necrosis by 5-aminolevulinic acid and 1,10-phenanthroline photodynamic therapy.Cancer Res. 1996; 56: 339-344PubMed Google Scholar) on ALA-induced fluorescence in vivo. Experiments were performed using male Syrian golden hamsters (60–80 g body weight) fitted with titanium dorsal skinfold chambers (n = 82). The animals were housed in single cages and had free access to food and water. Twenty-four hours after chamber preparation 2 × 105 cells of the amelanotic melanoma of the hamster (A-Mel-3) were implanted into each chamber when they did not show any macroscopic sign of inflammation (for details see:Endrich et al., 1980Endrich B. Asaishi K. Goetz A.E. Messmer K. Technical report. A new chamber technique for microvascular studies in unanaesthetized hamsters.Res Exp Med. 1980; 177: 125-134Crossref PubMed Scopus (359) Google Scholar;Asaishi et al., 1981Asaishi K. Endrich B. Goetz A.E. Messmer K. Quantitative analysis of microvascular structure and function in the amelanotic melanoma A-Mel-3.Cancer Res. 1981; 41: 1998-2004Google Scholar). The host tissue consists of a thin striated skin muscle and underlying subcutaneous adipose tissue, dermis, and epidermis (Abels et al., 1994Abels C. Heil P. Dellian M. Kuhnle G.E.H. Baumgartner R. Goetz A.E. In vivo kinetics and spectra of 5-aminolaevulinic acid induced fluorescence in an amelanotic melanoma of the hamster.Br J Cancer. 1994; 70: 826-833Crossref PubMed Scopus (77) Google Scholar). Six to 8 d later fluorescence microscopy and spectroscopy were performed, when a functioning tumor microcirculation was established (mean tumor diameter 4–6 mm). All surgical procedures were performed under pentobarbital anesthesia (50 mg per kg body weight intraperitoneal; Nembutal, Sanofi-LEVA, Hanover, Germany). The animals tolerated the chamber well and showed no signs of discomfort. ALA hydrochloride (Medac, Hamburg, Germany) was dissolved in distilled water and buffered to pH 7.4. The solution was prepared freshly in concentrations of 1%, 3%, or 10% at a temperature of 32°C. Before the topical application of ALA solution for 1 h, 4 h, or 8 h the animals were randomly assigned to each group. The ALA solution (100 μl) was applied to each preparation, which was then covered tightly using an adhesive foil (Opraflex, Lohmann GmbH, Neuwied, Germany) to avoid drying of the chamber. At the end of the application time ALA was removed and the chamber was rinsed using physiologic saline. For simultaneous application of ALA and glycine 3% ALA and an equimolar (20 μM) or a 10-fold higher concentration (200 μM) of glycine (Sigma, St Louis, MO) were prepared as described above and applied to the chamber. To reduce the iron concentration in the tissue desferrioxamine (1% or 3%) was added 1 h prior to and removed before the application of ALA (3%, 4 h). The tetrapyrrole modulator 1,10-phenanthroline (7.5 mM) was added simultaneously because it acts as an enhancer of porphyrin accumulation upon the exogenous application of ALA (3%, 4 h). The fluorescence intensity was recorded thereafter. For intravital fluorescence microscopy of tumor and surrounding host tissue the awake, chamber-bearing hamster was immobilized in a Perspex tube on a custom-made stage (Effenberger, Munich, Germany) under a Leitz microscope (Leitz, Munich, Germany, type 307-143003/51466) in the dark. For subtraction of autofluorescence, intravital microscopy was performed prior to the topical application of ALA. After application and removal of ALA the chamber was occluded with a new coverglass. At 1, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300, and 360 min and 24 h following application (p.a.) ALA-induced fluorescence was measured in tumor and surrounding host tissue. ALA-induced porphyrins were excited for 2 s with a power density of 200–300 μW per cm2 and at a wavelength of 355–425 nm (HBO mercury lamp, 100 W). Fluorescence was detected above 610 nm and recorded by a silicon-intensified target video camera (C 2400–08, Hamamatsu, Herrsching, Germany). Fluorescence images were acquired using a digital image analysis system and stored on a hard disk (IBAS 2000, Kontron, Eching, Germany). Spatial inhomogeneities of light or camera were compensated by shading correction. Tissue autofluorescence was subtracted and fluorescence intensity was measured densitometrically off-line. Fluorescence values are given in percentage of the solid fluorescence reference signal (Impregum F, Seefeld, Germany) inserted into each chamber. The geometric resolution of all images was 512 × 512 pixels by a densitometric resolution of 256 gray values. To determine ALA-induced fluorescence in tumor and surrounding host tissue, regions of interest (50 × 50 μm2) were chosen in a transillumination image prior to ALA application. These region of interest did not overlay larger blood vessels. Vertical cryostat sections did not show any fluorescence intensity gradient from the surface to the basis of the chamber indicating a rather homogeneous penetration of ALA in the tissue (data not shown). Fluorescence spectroscopy was performed using an intensified optical multichannel analyzer (O/SMA 3, Spectroscopy Instruments, Gilching, Germany) as described previously (Abels et al., 1994Abels C. Heil P. Dellian M. Kuhnle G.E.H. Baumgartner R. Goetz A.E. In vivo kinetics and spectra of 5-aminolaevulinic acid induced fluorescence in an amelanotic melanoma of the hamster.Br J Cancer. 1994; 70: 826-833Crossref PubMed Scopus (77) Google Scholar). Fluorescence emission spectra were determined in vivo from tumor and surrounding host tissue shortly after 4 h of topical application of 3% ALA. Statistical analysis of the data was performed using the paired-t-test for related samples (Sigma Stat-Computer program, Jandel Scientific, Erkrath, Germany). Differences were considered significant if p < 0.05. The fluorescence kinetics of ALA-induced porphyrins are shown in Figure 1,Figure 2,Figure 3. After 1 h application of ALA all groups showed a similar increase of the fluorescence intensity in the tumor and the surrounding host tissue, which was faster in the tumor as compared with the surrounding host tissue (Figure 1). Maxima in tumor (1% ALA, 94% ± 16%; 3% ALA, 129% ± 26%; 10% ALA, 117% ± 23%) were measured 150 min after end of application (1 h). After application for 4 h (Figure 2) the maximal fluorescence intensity in the tumor was measured 15 min p.a. (1% ALA, 80% ± 16%), 60 min p.a. (3% ALA, 215% ± 47%), or 15 min p.a. (10% ALA, 195% ± 81%). A concentration of 10% ALA did not increase the maximal fluorescence intensity as compared with 3% ALA following 4 h application, whereas an application of 4 h yielded a significantly higher fluorescence intensity as compared with 1 h using either 3% or 10% ALA. The maximal fluorescence intensity following 8 h application was measured immediately thereafter for all concentrations (1% ALA, 26% ± 7%; 3% ALA, 174% ± 34%; 10% ALA, 228% ± 42%) (Figure 3). Interestingly, the fluorescence intensity of the surrounding host tissue exceeded that measured in tumors after 8 h application and 1% ALA over the entire observation period. In all other groups the fluorescence intensity was always significantly higher in tumor – at least during the first hour after application. Moreover, there was no statistically significant difference of the fluorescence intensity measured in the surrounding host tissue regarding application time or concentration. Twenty-four hours after topical application hardly any fluorescence was detectable in all groups neither in tumor nor in surrounding host tissue.Figure 2Fluorescence kinetics of endogenously formed porphyrins in tumor and host tissue after topical application of 1%, 3%, and 10% ALA solution for 4 h. Mean ± SEM; *p < 0.05, tumor versus host tissue; n = 6.View Large Image Figure ViewerDownload (PPT)Figure 3Fluorescence kinetics of endogenously formed porphyrins in tumor and host tissue after topical application of 1%, 3%, and 10% ALA solution for 8 h. Mean ± SEM; *p < 0.05, tumor versus host tissue; n = 6.View Large Image Figure ViewerDownload (PPT) For optimal photodynamic therapy using this tumor model the tumor/host tissue fluorescence ratio was calculated for the different groups. Maxima of each group are presented in Table 1. The highest ratios were found already after 1 h of application independent from the concentration applied (1% ALA, 22:1; 3% ALA, 30:1; 10% ALA, 30:1). By increasing the application time (4 h and 8 h) the ratio between tumor and surrounding tissue could not be improved. Moreover, there is a tendency that the fluorescence ratio as indicator of the selectivity of ALA-induced porphyrins is decreasing with increasing application time.Table 1Maximal tumor/host tissue fluorescence ratio (time after application)Application timeALA 1%ALA 3%ALA 10%1 h22:1 (60 min)30:1 (15 min)30 : 1 (1 min)4 h5:1 (60 min)10:1 (30 min)10 : 1 (15 min)8 h0.7:1 (15 min)4:1 (1 min)16 : 1 (15 min) Open table in a new tab Emission spectra of the tumor and surrounding host tissue were registered 30 min following 4 h application and 3% ALA, at the time the maximal fluorescence ratio was found for this group. The emission bands show maxima at 637 and 704 nm in tumor as well as host tissue (spectra not shown). These peaks indicate the presence of PPIX. The simultaneous application of ALA (3%) and the amino acid glycine (20 μM = equimolar concentration) reduced the ALA-induced fluorescence (36%) significantly in tumor as compared with ALA alone (100%) (Figure 4b). By increasing the glycine concentration (200 μM) the induction of fluorescence by ALA (22%) was further significantly reduced (Figure 4c). The intensity of the ALA-induced fluorescence in the host tissue was also affected but to a lesser degree. Thus, the simultaneous application of ALA and the amino acid glycine interferes with the formation of porphyrins in tumor and surrounding host tissue. The fluorescence intensity in the surrounding normal tissue increased following application of desferrioxamine (3%) and there was no statistically significant difference as compared with the tumor (Figure 5a, b). Interestingly, the preincubation with desferrioxamine in a concentration of either 1% or 3% reduced significantly the ALA-induced fluorescence in the tumor. Moreover, adding the tetrapyrrole modulator 1,10-phenanthroline and ALA (3%) an increase of the fluorescence intensity as compared with ALA alone was not observed neither in tumor nor surrounding host tissue (Figure 5c). Intravital fluorescence microscopy and spectroscopy of the amelanotic melanoma implanted in the dorsal skinfold chamber can be used successfully to determine the kinetics of intravenously applied photosensitizers, which provide the basis for optimal fluorescence diagnosis or photodynamic therapy in this model (Leunig et al., 1993Leunig M. Richert C. Gamarra F. Lumper W. Vogel E. Jocham D. Goetz A.E. Tumor localisation kinetics of photofrin and three synthetic porphyrinoids in an amelanotic melanoma of the hamster.Br J Cancer. 1993; 68: 225-234Crossref PubMed Scopus (40) Google Scholar;Abels et al., 1994Abels C. Heil P. Dellian M. Kuhnle G.E.H. Baumgartner R. Goetz A.E. In vivo kinetics and spectra of 5-aminolaevulinic acid induced fluorescence in an amelanotic melanoma of the hamster.Br J Cancer. 1994; 70: 826-833Crossref PubMed Scopus (77) Google Scholar). In the current investigation we used this model for the respective analysis following topical application of ALA and to elucidate the so far unknown mechanism of selectivity of the ALA-induced porphyrins, as previous experiments did not reveal a reduced activity of the ferrochelatase in this model (Abels et al., 1994Abels C. Heil P. Dellian M. Kuhnle G.E.H. Baumgartner R. Goetz A.E. In vivo kinetics and spectra of 5-aminolaevulinic acid induced fluorescence in an amelanotic melanoma of the hamster.Br J Cancer. 1994; 70: 826-833Crossref PubMed Scopus (77) Google Scholar). The concentrations administered for this investigation were chosen according to topically applied doses for fluorescence diagnosis of bladder tumors (3% ALA) (Kriegmair et al., 1994Kriegmair M. Baumgartner R. Knüchel R. et al.Photodynamische Diagnose urothelialer Neoplasien nach intravesikaler Instillation von 5-Aminolävulinsäure.Urologie. 1994; 33: 270-275PubMed Google Scholar) or for photodynamic therapy of skin tumors (10% ALA) (Szeimies et al., 1995Szeimies R.M. Karrer S. Sauerwald A. Landthaler M. Photodynamic therapy with topical application of 5-aminolevulinic acid in the treatment of actinic keratosis: an initial clinical study.Dermatology. 1995; 192: 246-251Crossref Scopus (131) Google Scholar). The maximal fluorescence intensity was found shortly after 4 h application of 3% ALA, which did not increase by adding a higher concentration (10%) or by longer application (8 h) indicating a saturation of the intracellular uptake or porphyrin formation in the amelanotic melanoma (Figure 1,Figure 2,Figure 3). This finding is in accordance with an earlier study using this model following intravenous administration of ALA (Abels et al., 1994Abels C. Heil P. Dellian M. Kuhnle G.E.H. Baumgartner R. Goetz A.E. In vivo kinetics and spectra of 5-aminolaevulinic acid induced fluorescence in an amelanotic melanoma of the hamster.Br J Cancer. 1994; 70: 826-833Crossref PubMed Scopus (77) Google Scholar). The increase of fluorescence in tumor is faster as compared with surrounding host tissue following topical application indicating a faster uptake or increased heme biosynthesis and not a reduced activity of the ferrochelatase – a similar activity of the ferrochelatase in the amelanotic melanoma as compared with skin was shown recently (Fritsch et al., 1998Fritsch C. Goerz G. Ruzicka T. Photodynamic therapy in dermatology.Arch-Dermatol. 1998; 134: 207-214Crossref PubMed Scopus (261) Google Scholar). A faster increase was also observed in actinic keratoses and squamous cell carcinomas of mice as compared with normal skin (Van der Veen et al., 1996Van der Veen N. de Bruijn H.S. Berg R.J.W. Star W.M. Kinetics and localisation of PpIX fluorescence after topical and systemic ALA application observed in skin tumors of UVB-treated mice.Br J Cancer. 1996; 73: 925-930Crossref PubMed Scopus (52) Google Scholar). In that study, however, only a single concentration (20%) and a single application time (4 h) were studied (Van der Veen et al., 1996Van der Veen N. de Bruijn H.S. Berg R.J.W. Star W.M. Kinetics and localisation of PpIX fluorescence after topical and systemic ALA application observed in skin tumors of UVB-treated mice.Br J Cancer. 1996; 73: 925-930Crossref PubMed Scopus (52) Google Scholar). The maximal fluorescence intensity was measured 6 h after the end of the 4 h application time of ALA, whereas in our study it was found already 3 h after the 1 h application and 30 min after the 4 h application. This difference can be explained by the lack of an overlying stratum corneum in our model. Thus, it is not surprising that a similar time course was measured in bladder tumors following 4 h of intravesical instillation and a decrease following 6 h of instillation (Xiao et al., 1998Xiao Z. Miller G.G. McCallum T.J. Brown- K.M. Lown J.W. Tulip J. Moore R.B. Biodistribution of Photofrin II and 5-aminolevulinic acid-induced protoporphyrin IX in normal rat bladder and bladder tumor models: implications for photodynamic therapy.Photochem Photobiol. 1998; 67: 573-583Crossref PubMed Scopus (30) Google Scholar). In the orthotopic bladder tumor model used, however, the selectivity of ALA-induced fluorescence was reduced following topical application as compared with intravenous injection. The fluorescence intensity in the amelanotic melanoma was 30 times higher (3% ALA, 1 h) (Table 1) as compared with surrounding host tissue exceeding the tumor/surrounding tissue ratios measured so far byVan der Veen et al., 1996Van der Veen N. de Bruijn H.S. Berg R.J.W. Star W.M. Kinetics and localisation of PpIX fluorescence after topical and systemic ALA application observed in skin tumors of UVB-treated mice.Br J Cancer. 1996; 73: 925-930Crossref PubMed Scopus (52) Google Scholar in squamous cell carcinomas and skin as well as byIinuma et al., 1995Iinuma S. Bachor R. Flotte T. Hasan T. Biodistribution and phototoxicity of 5-aminolevulinic acid-induced PpIX in an orthotopic rat bladder tumor model.J Urol. 1995; 153: 802-806Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar in a bladder tumor and surrounding urothelium. This is probably due to the underlying striated skin muscle in our model forming less porphyrins as tissue of epithelial origin. Nevertheless, it is noteworthy that in our model the selectivity of the induced porphyrins as indicated by the tumor/surrounding host tissue fluorescence ratio was decreasing with the longer application time (Table 1), which was similar to the orthotopic bladder tumor model (Xiao et al., 1998Xiao Z. Miller G.G. McCallum T.J. Brown- K.M. Lown J.W. Tulip J. Moore R.B. Biodistribution of Photofrin II and 5-aminolevulinic acid-induced protoporphyrin IX in normal rat bladder and bladder tumor models: implications for photodynamic therapy.Photochem Photobiol. 1998; 67: 573-583Crossref PubMed Scopus (30) Google Scholar). Thus, shorter application times of ALA seem to yield a better contrast between normal and neoplastic tissue due to the faster increase of the ALA-induced fluorescence in tumor as compared with the surrounding host tissue. This was also observed clinically by us in basal cell carcinomas and surrounding skin following topical application of 20% ALA in an W/O emulsion (Ackermann et al., 1998Ackermann G. Abels C. Bäumler W. Karrer S. Lang E. Landthaler M. Szeimies R.M. Fluorescence diagnosis of skin tumors following topical application of 5 aminolevulinic acid.J Invest Dermatol. 1998; 110: 677Google Scholar). Moreover, using a three-compartment model comparable rate coefficients were calculated for the amelanotic melanoma as well as for human basal cell carcinomas (unpublished data). Comparing the different routes of administration of ALA, either intravenously as investigated previously (Abels et al., 1994Abels C. Heil P. Dellian M. Kuhnle G.E.H. Baumgartner R. Goetz A.E. In vivo kinetics and spectra of 5-aminolaevulinic acid induced fluorescence in an amelanotic melanoma of the hamster.Br J Cancer. 1994; 70: 826-833Crossref PubMed Scopus (77) Google Scholar) or topically as in this study the kinetics after 1 h application of ALA are similar as compared with those after intravenous injection of 500 mg per kg. Moreover, the maximal fluorescence intensity of neoplastic tissue reached in this model is ≈190% following either topical or systemic administration. Taking these data into account a topical application might be more efficient as an uptake of ALA in other organs, e.g., liver, kidney, or a generalized photosensitivity and other systemic side-effects does not occur. The selectivity following topical application, however, might be slightly reduced as compared with the intravenous administration in our model, but not as marked as observed in an orthotopic bladder tumor model (Xiao et al., 1998Xiao Z. Miller G.G. McCallum T.J. Brown- K.M. Lown J.W. Tulip J. Moore R.B. Biodistribution of Photofrin II and 5-aminolevulinic acid-induced protoporphyrin IX in normal rat bladder and bladder tumor models: implications for photodynamic therapy.Photochem Photobiol. 1998; 67: 573-583Crossref PubMed Scopus (30) Google Scholar). The recording of the fluorescence spectra in tumor and surrounding host tissue revealed the characteristic emission bands of PPIX (Dailey and Smith, 1984Dailey H.A. Smith A. Differential interaction of porphyrins used in photoradiation therapy with ferrochelatase.Biochem J. 1984; 223: 441-445Crossref PubMed Scopus (133) Google Scholar;Bedwell et al., 1992Bedwell J. MacRobert A.J. Phillips D. Bown S.G. Fluorescence distribution and photodynamic effect of ALA-induced PP IX in the DMH rat colonic tumour model.Br J Cancer. 1992; 65: 818-824Crossref PubMed Scopus (252) Google Scholar). Additional peaks at 618 nm and 678 nm as seen after intravenous injection of ALA in this model (Abels et al., 1994Abels C. Heil P. Dellian M. Kuhnle G.E.H. Baumgartner R. Goetz A.E. In vivo kinetics and spectra of 5-aminolaevulinic acid induced fluorescence in an amelanotic melanoma of the hamster.Br J Cancer. 1994; 70: 826-833Crossref PubMed Scopus (77) Google Scholar) did not occur following topical application suggesting the formation of the porphyrin following intravenous injection emitting these bands elsewhere in the organism but not in tumor or surrounding host tissue. Selective ALA-induced accumulation of porphyrins is suggested to be a result of a reduced activity of the enzyme ferrochelatase as shown in hepatomas (Dailey and Smith, 1984Dailey H.A. Smith A. Differential interaction of porphyrins used in photoradiation therapy with ferrochelatase.Biochem J. 1984; 223: 441-445Crossref PubMed Scopus (133) Google Scholar) and colon carcinomas of rats (Van Hillersberg et al., 1992Van Hillersberg R. Van der Berg R. Kort J.W.O. Onno T.T. Wilson J.H.P. Selective accumulation of endogenously produced porphyrins in a liver metastasis model in rats.Gastroenterology. 1992; 103: 647-651PubMed Google Scholar). This enzymatic defect and also reduced intracellular iron levels (Rittenhouse-Diakun et al., 1995Rittenhouse-Diakun K. Van Leengoed H. Morgan J. Hryhorenko E. Paskiewics G. Whitaker J.E. Oseroff A.R. The role of transferrin receptor (CD71) in photodynamic therapy of activated and malignant lymphocytes using the heme precursor δ-aminolaevulinic acid (ALA).Photochem Photobiol. 1995; 61: 523-528Crossref PubMed Scopus (116) Google Scholar) are discussed as mechanisms for the selective fluorescence in the tumor as compared with surrounding host tissue. As indicated by the faster and higher porphyrin accumulation in our model following either intravenous or topical application the selective accumulation of porphyrins is mainly due to an increased uptake and not due to the reduced activity of the enzyme ferrochelatase (Abels et al., 1994Abels C. Heil P. Dellian M. Kuhnle G.E.H. Baumgartner R. Goetz A.E. In vivo kinetics and spectra of 5-aminolaevulinic acid induced fluorescence in an amelanotic melanoma of the hamster.Br J Cancer. 1994; 70: 826-833Crossref PubMed Scopus (77) Google Scholar,Abels et al., 1997Abels C. Fritsch C. Bolsen K. Szeimies R.M. Ruzicka T. Goerz G. Goetz A.E. Photodynamic therapy with 5-aminolaevulinic acid-induced porphyrins of an amelanotic melanoma.In Vivo. J Photochem Photobiol. 1997; 40: 76-83Crossref PubMed Scopus (35) Google Scholar;Fritsch et al., 1998Fritsch C. Goerz G. Ruzicka T. Photodynamic therapy in dermatology.Arch-Dermatol. 1998; 134: 207-214Crossref PubMed Scopus (261) Google Scholar). In addition, the fact that ALA-induced fluorescence could be saturated after systemic or topical application supports this hypothesis. Studies concerning the intracellular uptake of ALA were conducted in Saccharomyces cerevisiae indicating an active ALA uptake system in this eukaryotic model (Moretti et al., 1993Moretti M.B. Garcia S.R.C. Stella C. Ramos E.H. Battle A.M.C. δ-aminolaevulinic acid transport in Saccharomyces cerevisiae.Int J Biochem. 1993; 25: 1917-1924Crossref PubMed Scopus (25) Google Scholar,Moretti et al., 1995Moretti M.B. Garcia S.R.C. Chianelli M.S. Ramos E.H. Mattoon J.R. Battle A.M.C. Evidence that γ-aminobutyric acid and δ-aminolaevulinic acid share a common system into Sacharomyces cerevisiae.Cell Biol. 1995; 27: 169-173Google Scholar). This has already been confirmed in vitro using a melanoma cell line showing that ALA is transported via an active transport mechanism and not via diffusion (Kalka et al., 1997Kalka K. Fritsch C. Ruzicka T. Goerz G. Eckel J. δ-aminolaevulinic acid accumulates intrecellularly by active transport mechanisms and not via passive diffusion.Arch Dermatol Res. 1997; 289 (Suppl.): A1-A64PubMed Google Scholar). Major effects are not described on the uptake of ALA in mammalian cells by amino acids like γ-aminobutyric acid or glutamic acid with a similar chemical structure such as ALA (Washbrook et al., 1997Washbrook R. Fukuda H. Battle A. Riley P. Stimulation of tetrapyrrole synthesis in mammalian epithelial cells in culture by exposure to aminolaevulinic acid.Br J Cancer. 1997; 75: 381-387Crossref PubMed Scopus (16) Google Scholar). It has been shown, however, that the amino acid glycine given orally to patients suffering from acute porphyria increases the rate of excretion of ALA in their urine (Richards and Scott, 1961Richards F.F. Scott J.J. Glycine metabolism in acute porphyria.Clin Sci. 1961; 20: 387-400PubMed Google Scholar). Therefore, we assumed that the simultaneous application of ALA and glycine in equimolar concentration should reduce the ALA-induced fluorescence in our model. As supposed the simultaneous application of ALA and glycine yielded a reduction – probably concentration dependent – of ALA-induced fluorescence in tumor and surrounding host tissue (Figure 4). This finding suggests a glycine-mediated inhibition of the intracellular uptake of ALA into the amelanotic melanoma and to a significant lower degree into the surrounding host tissue in vivo. Knowing that this hypothesis has to be confirmed by the appropriate in vitro experiments we suggest that ALA and glycine seem to share the same mechanism of internalization into amelanotic melanoma cells. This finding is important regarding the unique selectivity of ALA-induced porphyrins in neoplastic tissue, in also making use of it for therapeutic strategies. To increase the accumulation and perhaps the selectivity of the ALA-induced porphyrins after topical application of ALA the iron chelator desferrioxamine (1% or 3%) was added 1 h prior to ALA. Desferrioxamine is a specific chelator for iron and may thus reduce the metabolization of PPIX into heme. Surprisingly, there was a decrease of the fluorescence intensity in the tumor following either concentration and the application of the higher concentration yielded even a slight increase of the fluorescence intensity in the surrounding tissue (Figure 5b) thus reducing the selectivity. This finding is in contrast to in vitro studies which showed an increased PPIX production in carcinoma cells using desferrioxamine and ALA (Ortel et al., 1993Ortel B. Tanew A. Hönigsmann H. Lethal photosensitization by endogenous porphyrins of PAM cells-modification by desferrioxamine.J Photochem Photobiol B. 1993; 17: 273-278Crossref PubMed Scopus (67) Google Scholar;Berg et al., 1996Berg K. Anholt H. Bech O. Moan J. The influence of iron chelators on the accumulation of protoporphyrin IX in 5-aminolaevulinic acid-treated cells.Br J Cancer. 1996; 74: 688-697Crossref PubMed Scopus (121) Google Scholar;Tan et al., 1997Tan W.C. Krasner N. O’Toole P. Lombard M. Enhancement of photodynamic therapy in gastric cancer cells by removal of iron.Gut. 1997; 41: 14-18Crossref PubMed Scopus (20) Google Scholar). Similar to those findingsPeng et al., 1996Peng Q. Moan J. Iani V. Nesland J.M. Effect of desferrioxamine on porduction of Ala-induced protoporphyrin IX in normal mouse skin.Proc SPIE. 1996; 2625: 51-57Crossref Google Scholar observed an enhanced PPIX formation and the iron chelator CP94 given intraperitoneally to rats seems also to be effective in increasing ALA-induced PPIX in the bladder as compared with ALA alone (Chang et al., 1997Chang S.C. MacRobert A.J. Porter J.B. Bown- S.G. The efficacy of an iron chelator (CP94) in increasing cellular protoporphyrin IX following intravesical 5-aminolaevulinic acid administration: an in vivo study.J Photochem Photobiol: B. 1997; 38: 114-122Crossref PubMed Scopus (44) Google Scholar). In these in vivo studies, however, only normal tissue, normal mouse skin, and normal rat urothelium, respectively, were investigated. And this finding is in accordance with our results as there was also an increase of ALA-induced fluorescence in the surrounding normal tissue following application of desferrioxamine (3%) (Figure 5b). This pronounced increase in the surrounding tissue reduced the selectivity of ALA-induced fluorescence following application of desferrioxamine in vivo as already speculated byBerg et al., 1996Berg K. Anholt H. Bech O. Moan J. The influence of iron chelators on the accumulation of protoporphyrin IX in 5-aminolaevulinic acid-treated cells.Br J Cancer. 1996; 74: 688-697Crossref PubMed Scopus (121) Google Scholar according to their in vitro findings. As reported byRebeiz et al., 1996Rebeiz N. Arkins S. Rebeiz C.A. Simon J. Zachary J.F. Kelley K.W. Induction of tumor necrosis by 5-aminolevulinic acid and 1,10-phenanthroline photodynamic therapy.Cancer Res. 1996; 56: 339-344PubMed Google Scholar the simultaneous injection of 1,10-phenanthroline (7.5 mM) yielded a higher porphyrin accumulation in a mice fibrosarcoma model following intratumoral application of ALA. As shown in Figure 5(c) there was no effect on ALA-induced fluorescence when applied topically neither in the tumor nor in the surrounding host tissue in our model. As it is supposed that 1,10-phenanthroline interacts rather unspecifically with enzymes of the heme biosynthesis due to its chemical structure, we also expected an increase of the ALA-induced fluorescence during the observation time of 6 h (Rebeiz et al., 1996Rebeiz N. Arkins S. Rebeiz C.A. Simon J. Zachary J.F. Kelley K.W. Induction of tumor necrosis by 5-aminolevulinic acid and 1,10-phenanthroline photodynamic therapy.Cancer Res. 1996; 56: 339-344PubMed Google Scholar). In the study by Rebeiz et al. it was already noticed that the accumulation of PPIX after application of ALA/1,10-phenanthroline was significantly higher in single cell suspensions (7-fold increase versus ALA alone) as compared with solid tumors (2-fold increase versus ALA alone). Thus, the increase of ALA-induced fluorescence in the amelanotic melanoma due to the addition of 1,10-phenanthroline might be not significant enough to be detected by our set up. To elucidate the underlying mechanisms resulting in the unique selectivity of ALA-induced fluorescence the intracellular uptake of ALA should be characterized and further investigations in vivo should focus on whether the faster and higher accumulation of porphyrins in tumors is due to an increased ALA uptake or just reflects an increased porphyrin formation due to a higher demand of tumors for heme. The authors gratefully acknowledge the critical comments on this manuscript by Prof. K. Messmer, Director of the Institute for Surgical Research, Ludwig-Maximilians-Universität, Munich, Germany. The investigation was supported by a grant of the Bundesministerium für Bildung und Forschung to A.E.G. (grant no. 0706903A5)" @default.
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