Showing papers on "Protoporphyrin IX published in 2000"
TL;DR: The influence of parameters such as lipophilicity, concentration, time, and pH value on PpIX formation induced by ALA and its esters is investigated in human cell lines originating from the lung and bladder, finding ALA esters are found to be more lipophilic than the free acid.
Abstract: Protoporphyrin IX (PpIX) is used as a fluorescence marker and photosensitizing agent in photodynamic therapy (PDT). A temporary increase of PpIX in tissues can be obtained by administration of 5-aminolevulinic acid (ALA). Lipophilicity is one of the key parameters defining the bioavailability of a topically applied drug. In the present work, octanol-water partition coefficients of ALA and several of its esters have been determined to obtain a parameter related to their lipophilicity. The influence of parameters such as lipophilicity, concentration, time, and pH value on PpIX formation induced by ALA and its esters is then investigated in human cell lines originating from the lung and bladder. ALA esters are found to be more lipophilic than the free acid. The optimal concentration (c(opt), precursor concentration at which maximal PpIX accumulation is observed) is then measured for each precursor. Long-chained ALA esters are found to decrease the c(opt) value by up to two orders of magnitude as compared to ALA. The reduction of PpIX formation observed at higher concentrations than c(opt) is correlated to reduced cell viability as determined by measuring the mitochondrial activity. Under optimal conditions, the PpIX formation rate induced by the longer-chained esters is higher than that of ALA or the shorter-chained esters. A biphasic pH dependence on PpIX generation is observed for ALA and its derivatives. Maximal PpIX formation is measured under physiological conditions (pH 7.0-7.6), indicating that further enhancement of intracellular PpIX content may be achieved by adjusting the pharmaceutical formulation of ALA or its derivatives to these pH levels.
219 citations
TL;DR: Intercompartmental signaling that synchronizes the activities of the first steps in tetrapyrrolic metabolism with the late steps for the synthesis of end products is discussed.
Abstract: Magnesium-protoporphyrin IX chelatase (Mg-chelatase) is located at the branchpoint of tetrapyrrole biosynthesis, at which point protoporphyrin IX is distributed for the synthesis of chlorophyll and heme. We investigated the regulatory contribution of Mg-chelatase to the flow of metabolites. In plants, the enzyme complex consists of three subunits, designated CHL D, CHL I, and CHL H. Transgenic tobacco (Nicotiana tabacum) plants expressing antisense RNA for the Mg-chelatase subunit CHL H were analyzed to elucidate further the role of Mg-chelatase in the distribution of protoporphyrin IX into the branched tetrapyrrolic pathway. The transgenic plants displayed a reduced growth rate and chlorophyll deficiency. Both phenotypical properties were correlated with lower Mg-chelatase activity. Unexpectedly, less protoporphyrin IX and heme accumulated, and a decrease in 5-aminolevulinate (ALA)-synthesizing capacity and ALA dehydratase activity paralleled the progressive reduction in Mg-chelatase activity in the transformants compared with control plants. The reduced activities of the early enzymatic steps corresponded with lower levels of transcripts encoding glutamyl-tRNA reductase and ALA-dehydratase. The decreased expression and activities of early enzymes in the pathway could be explained by a feedback-controlled mechanism in response to lower Mg-chelatase activity. We discuss intercompartmental signaling that synchronizes the activities of the first steps in tetrapyrrolic metabolism with the late steps for the synthesis of end products.
166 citations
TL;DR: An essential role for light-induced Mg-protoporphyrin IX accumulation in this chloroplast-to-nucleus signalling pathway is suggested which makes this plastidic compound accessible to the cytoplasm/nucleu where the downstream signalling pathway may be activated.
Abstract: Chlorophyll precursors Mg-protoporphyrin IX and its monomethylester are candidates for plastid-derived molecules involved in light signalling from the chloroplast to the nucleus. The pool sizes of these two Mg2+-containing porphyrins and of protoporphyrin IX transiently increased upon a shift of Chlamydomonas cultures from dark to light. This increase coincided with the accumulation of mRNAs encoded by the nuclear genes HSP70A and HSP70B. Analysis of a mutant (brs-1), previously shown to be defective in the light induction of these genes, revealed high levels of protoporphyrin IX but no light-induced increase in the levels of Mg2+-containing porphyrins. Inhibitors of cytoplasmic protein synthesis prevented both the light-induced rise in pool levels and induction of the HSP70 genes. Similarly, pre-gametes, intermediates of sexual differentiation, lacked both responses to light. The block in light induction of the HSP70 genes in inhibitor-treated cells and in pre-gametes could be circumvented by the exogenous addition of Mg-protoporphyrin IX in the dark. This suggests an essential role for light-induced Mg-protoporphyrin IX accumulation in this chloroplast-to-nucleus signalling pathway. However, accumulation of this porphyrin in the dark - presumably in the chloroplast - did not result in induction. A second crucial role for light in this signalling pathway is postulated which makes this plastidic compound accessible to the cytoplasm/nucleus where the downstream signalling pathway may be activated.
159 citations
TL;DR: Results obtained indicate that association of 5-ALA, EDTA and 20% DMSO may enhance the delivery of 4-aminolevulinic acid to the skin in the topical PDT, and this chelator could protect 5- ALA from decomposition during prolonged topical administration.
129 citations
TL;DR: In this paper, the authors present quantitative fluorescence measurements and results in the visualization of cancerous oral mucosa with 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX).
Abstract: Objectives: Early cancer detection is the best way to improve the prognosis of patients with oral cancer. Therefore this study presents quantitative fluorescence measurements and results in the visualization of cancerous oral mucosa with 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX).
Methods: Time progression and type of porphyrin accumulation were analyzed in neoplastic and surrounding healthy tissue of 58 patients with a suspected cancer of the oral cavity by measuring emission spectra of 5-ALA-induced PPIX fluorescence. Fluorescence images in the red and green spectral range from the tumor tissue were recorded with a charge-coupled device camera.
Results: After topical application of 0.4% 5-ALA and incubation for 1 to 2.5 hours, all patients revealed higher intensities of red fluorescence in neoplastic tissue compared with the surrounding normal tissue. Maximum contrast was reached after 1.5 hours of incubation. In 13.8% (n = 8) of the patients, additional findings like dysplasia, carcinoma in situ, primary tumor, secondary carcinomas, and tumor branches were found by means of fluorescence marking in contrast to white light examination. An evaluation of the biopsy specimens resulted in a specificity of 60% and a sensitivity of 99%.
Conclusions: As a fluorescent marker, PPIX could represent a possible new diagnostic tool to detect early malignant and secondary lesions in the oral cavity. In addition, 5-ALA-induced PPIX fluorescence is promising as a useful intraoperative tool for determining adequate surgical margins of resection. Further investigations aim to assess this diagnostic procedure as a sensitive and clinically reliable method for patients with oral cancer.
119 citations
TL;DR: Overproduction of protoporphyrinogen oxidase neutralizes the herbicidal action, prevents the accumulation of the substrate protoporalinogen IX, and consequently abolishes the light-dependent phytotoxicity of acifluorfen.
Abstract: The use of herbicides to control undesirable vegetation has become a universal practice. For the broad application of herbicides the risk of damage to crop plants has to be limited. We introduced a gene into the genome of tobacco (Nicotiana tabacum) plants encoding the plastid-located protoporphyrinogen oxidase of Arabidopsis, the last enzyme of the common tetrapyrrole biosynthetic pathway, under the control of the cauliflower mosaic virus 35S promoter. The transformants were screened for low protoporphyrin IX accumulation upon treatment with the diphenyl ether-type herbicide acifluorfen. Leaf disc incubation and foliar spraying with acifluorfen indicated the lower susceptibility of the transformants against the herbicide. The resistance to acifluorfen is conferred by overexpression of the plastidic isoform of protoporphyrinogen oxidase. The in vitro activity of this enzyme extracted from plastids of selected transgenic lines was at least five times higher than the control activity. Herbicide treatment that is normally inhibitory to protoporphyrinogen IX oxidase did not significantly impair the catalytic reaction in transgenic plants and, therefore, did not cause photodynamic damage in leaves. Therefore, overproduction of protoporphyrinogen oxidase neutralizes the herbicidal action, prevents the accumulation of the substrate protoporphyrinogen IX, and consequently abolishes the light-dependent phytotoxicity of acifluorfen.
118 citations
TL;DR: The expression and activity of several tetrapyrrolic enzymes were reduced in parallel to lower the Mg-chelatase activity, and the rate-limiting synthesis of 5-aminolevulinate was also decreased in the transgenic lines analyzed.
Abstract: The chelation of Fe2+ and Mg2+ ions forms protoheme IX and Mg-protoporphyrin IX, respectively, and the latter is an intermediate in chlorophyll synthesis. Active magnesium protoporphyrin IX chelatase (Mg-chelatase) is an enzyme complex consisting of three different subunits. To investigate the function of the CHL I subunit of Mg-chelatase and the effects of modified Mg-chelatase activity on the tetrapyrrole biosynthetic pathway, we characterized N. tabacum transformants carrying gene constructs with the Chl I cDNA sequence in antisense and sense orientation under the control of the CaMV 35S promoter. Both elevated and diminished levels of Chl I mRNA and Chl I protein led to reduced Mg-chelatase activities, reflecting a perturbation of the assembly of the enzyme complex. The transformed plants did not accumulate the substrate of Mg-chelatase, protoporphyrin IX, but the leaves contained less chlorophyll and possessed increased chlorophyll a/b ratios, as well as a deficiency of light-harvesting chlorophyll binding proteins of photosystems I and II. The expression and activity of several tetrapyrrolic enzymes were reduced in parallel to lower the Mg-chelatase activity. Consistent with the lower chlorophyll contents, the rate-limiting synthesis of 5-aminolevulinate was also decreased in the transgenic lines analyzed. The consequence of reduced Mg-chelatase on early and late steps of chlorophyll synthesis, and on the organization of light harvesting complexes is discussed.
117 citations
TL;DR: The in vitro culture of urothelial cells and fibroblasts indicates that the most important metabolic step for PPIX accumulation in the urinary bladder is the transition from PPIX to heme, which does support the validation of photodynamic diagnosis and might also lead the way to a highly specific tumor related molecule.
Abstract: 5-Aminolevulinic acid (ALA)–supported fluorescence endoscopy of the urinary bladder results in a detection rate of bladder cancer superior to that of white light endoscopy. The different accumulation of the metabolite protoporphyrin IX (PPIX) in tumor cells after ALA instillation is poorly understood; however, it is crucial to optimize diagnosis and potential phototherapy. For systematic analysis of cell-type specific PPIX accumulation and metabolism two human bladder carcinoma cell lines (RT4 and J82), a normal urothelial cell line (UROtsa), and a fibroblast cell line (N1) were chosen, and grown in two different growth states to model important tissue components of the urinary bladder, i.e. tumor, normal epithelium and stroma. To quantitate PPIX content, fluorescence intensities measured by flow cytometry were matched with cellular PPIX extraction values, and related to relative ferrochelatase activity, cellular iron content, number of transferrin receptors per cell and porphobilinogen deaminase (PBGD) activity. For in vitro experiments, the initial correlation of relative flow cytometric and spectrometric measurements of PPIX provides a calibration curve for consequent flow cytometric PPIX quantification. Lower fluorescence of normal cells could be explained by significant differences of ferrochelatase activity and iron content in comparison to tumor cells. However, the content of iron was not related to transferrin receptor content. PBGD activity seemed to play a minor role for the differential accumulation of PPIX in urothelial cells. In conclusion, the in vitro culture of urothelial cells and fibroblasts indicates that the most important metabolic step for PPIX accumulation in the urinary bladder is the transition from PPIX to heme. Further investigation of PPIX metabolism does support the validation of photodynamic diagnosis, and might also lead the way to a highly specific tumor related molecule.
114 citations
TL;DR: The X-ray structure revealed the active site of the enzyme, to which only one of the isomers was bound, and for the first time allowed characterization of the mode of porphyrin macrocycle distortion by ferrochelatase.
109 citations
TL;DR: It is determined that the photochemistry subsequent to TPE is very similar to that found for one‐photon excitation, and the photoproducts observed possess very intense TPE fluorescence spectra, which allows their detection at low relative concentrations.
Abstract: The spectroscopy and photochemistry of protoporphyrin IX in ethanol and in Triton X-100 micelle solution have been examined using near-infrared two-photon excitation (TPE). TPE will allow photodynamic therapy with highly localized light dosage. We have determined that the photochemistry subsequent to TPE is very similar to that found for one-photon excitation. Moreover, the photoproducts observed possess very intense TPE fluorescence spectra, which allows their detection at low relative concentrations.
108 citations
TL;DR: ALA- and ALAHE-induced PpIX fluorescence kinetics are compared in the normal nude mouse skin, and the results indicates that ALA HE diffuses more slowly across the stratum corneum than ALA.
Abstract: An important limitation of topical 5-aminolevulinic acid (ALA)-based photodetection and photodynamic therapy is that the amount of the fluorescing and photosensitizing product protoporphyrin IX (PpIX) formed is limited. The reason for this is probably the limited diffusion of ALA through the stratum corneum. A solution to this problem might be found in the use of ALA derivatives, as these compounds are more lipophilic and therefore might have better penetration properties than ALA itself. Previous studies have shown that ALA hexyl ester (ALAHE) is more successful than ALA for photodetection of early (pre)malignant lesions in the bladder. However, ALA pentyl ester slightly increased the in vivo PpIX fluorescence in early (pre)malignant lesions in hairless mouse skin compared to ALA. The increased PpIX fluorescence is located in the stratum corneum and not in the dysplastic epidermal layer. In the present study, ALA- and ALAHE-induced PpIX fluorescence kinetics are compared in the normal nude mouse skin, of which the permeability properties differ from the bladder. Application times and ALA(HE) concentrations were varied, the effect of a penetration enhancer and the effect of tape stripping the skin before or after application were investigated. Only during application for 24 h, did ALAHE induce slightly more PpIX fluorescence than ALA. After application times ranging from 1 to 60 min, ALA-induced PpIX fluorescence was higher than ALAHE-induced PpIX fluorescence. ALA also induced higher PpIX production than ALAHE after 10 min of application with concentrations ranging from 0.5 to 40%. The results of experiments with the penetration enhancer and tape stripping indicated that the stratum corneum acts a barrier against ALA and ALAHE. Use of penetration enhancer or tape stripping enhanced the PpIX production more in the case of ALAHE application than in the case of ALA application. This, together with the results from the different application times and concentrations indicates that ALAHE diffuses more slowly across the stratum corneum than ALA.
TL;DR: The results suggest that at this time point, a minimal and fairly localized apoptotic effect is produced in brain tissues, the extent of which depends largely on the particular photosensitizer.
Abstract: The apoptotic response of normal brain and intracranial VX2 tumour following photodynamic therapy (PDT) mediated by 5 different photosensitizers (Photofrin, 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX), chloroaluminium phthalocyanine (AlCIPc), Tin Ethyl Etiopurpurin (SnET(2)), and meta -tetra(hydroxyphenyl)chlorin (m THPC)) was evaluated following a previous analysis which investigated the necrotic tissue response to PDT at 24 h post treatment. Free DNA ends, produced by internucleosomal DNA cleavage in apoptotic cells, were stained using a TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling) assay. Confocal laser scanning microscopy (CLSM) was used to quantify the local incidence of apoptosis and determine its spatial distribution throughout the brain. The incidence of apoptosis was confirmed by histopathology, which demonstrated cell shrinkage, pyknosis and karyorrhexis. At 24 h post PDT, AlClPc did not cause any detectable apoptosis, while the other photosensitizers produced varying numbers of apoptotic cells near the region of coagulative necrosis. The apoptotic response did not appear to be related to photosensitizer dose. These results suggest that at this time point, a minimal and fairly localized apoptotic effect is produced in brain tissues, the extent of which depends largely on the particular photosensitizer.
TL;DR: Human adenocarcinoma cells of the line WiDr were incubated with 5-aminolevulinic acid to induce protoporphyrin IX (PpIX) and exposed to laser light of wavelength 635 nm, and the PpIX fluorescence decreased with increasing exposure.
Abstract: Human adenocarcinoma cells of the line WiDr were incubated with 5-aminolevulinic acid to induce protoporphyrin IX (PpIX) and then exposed to laser light of wavelength 635 nm. The PpIX fluorescence decreased with increasing exposure. The decay rate was slightly dependent on the initial PpIX concentration. The PpIX fluorescence was halved by a fluence of about 40 J/cm2. Several fluorescing photoproducts were formed. The main one, supposedly the chlorine-type photoprotoporphyrin (Ppp), had a fluorescence excitation spectrum stretching out to about 680 nm with a maximum at around 668 nm. The formation kinetics of this product was dependent on the initial PpIX concentration. Moreover, it was selectively bleached by exposure to light at 670 nm. A photoproduct with an emission maximum at 652 nm, different from Ppp, remained after this exposure. Traces of a photoproduct(s) with fluorescence emission slightly blue-shifted compared with that of PpIX, supposedly water-soluble porphyrins, were also detected after light exposure.
Journal Article•
TL;DR: Results suggest the treatment with red light can be potentially employed as an therapeutic method to inactivate certain pathogenic strains of porphyrin producing bacteria without the use of external photosensitizers.
Abstract: With the increase in the number of antibiotic resistant strains of microorganism, the search for alternative treatments of microbial infections becomes all the more important. We report a novel method for bacterial inactivation based on the optical excitation of the naturally occurring (endogenous) photosensitzing porphyrins by red light. In particular, the pathogenic Gram-positive porphyrin producing ATCC strains Propionibacterium acnes, Actinomyces odontolyticus and Porphyromonas gingivalis were investigated. Sensitive autofluorescence spectroscopy revealed that these bacteria naturally synthezise the fluorescent photosensitizer protoporphyrin IX. In addition, bacterial plaque samples of periodontitis patients were studied. Non-labeled fluorescent bacterial colonies were exposed to red light at 632.8 nm, 100 mW/cm2 light intensity and 360 J/cm2 energy density using a helium-neon laser. The survival rate after a single phototreatment with red light was found to be 0.58 +/- 0.09 in the case of Propionibacterium acnes, 0.30 +/- 0.04 in Actinomyces odontolyticus and 0.59 +/- 0.10 in Porphyrormonas gingivalis compared to non-exposed bacteria suspensions. No photoeffect was found for the bacterium Streptococcus mutans which exhibited no detectable porphyrin autofluorescence. Red-light exposed plaque samples of patients showed significant reduction of colony forming units by 50% as well as a pronounced photoeffect on the pigmented species Prevotella intermedia. Taken together, these results suggest the treatment with red light can be potentially employed as an therapeutic method to inactivate certain pathogenic strains of porphyrin producing bacteria without the use of external photosensitizers.
TL;DR: It is found that homozygous drc(m248) mutant embryos have a G-->T transversion at a splice donor site in the ferrochelatase gene, creating a premature stop codon, suggesting a deficiency in the activity of ferroChelatase, the terminal enzyme in the pathway for heme biosynthesis.
TL;DR: Overall, the presently available data indicate that the risk for secondary skin carcinoma after topical ALA-PDT seems to be low, but further studies must be carried out to evaluate the carcinogenic risk of ALA -PDT in conditions predisposed to skin cancer.
TL;DR: A porphyrin covalently appended monolayer film on a glass substrate prepared by axial coordination reaction of protoporphrin IX zinc (ZnPP) and a self-assembled monolayers of (3-aminopropyl)trimeth is described in this paper.
Abstract: A porphyrin covalently appended monolayer film on a glass substrate prepared by axial coordination reaction of protoporphyrin IX zinc (ZnPP) and a self-assembled monolayer of (3-aminopropyl)trimeth...
TL;DR: Ethylene glycol esters and amino acid pseudodipeptide derivatives of ALA are synthesized and characterized as potential specific substrates for cellular esterases and aminopeptidases, respectively and shown to be useful at concentrations below their cytotoxicity threshold.
Abstract: Protoporphyrin IX (PpIX) is used as a photosensitizing agent in photodynamic detection and therapy (PDT) of cancer and is synthesized intracellularly from aminolevulinic acid (ALA) precursors. To evaluate means to specifically target ALA derivatives to defined cells, we have synthesized and characterized ethylene glycol esters and amino acid pseudodipeptide derivatives of ALA as potential specific substrates for cellular esterases and aminopeptidases, respectively. The PpIX formation induced by these products was investigated using cultures of human and rat cell lines of carcinoma and endothelial origins. The cytotoxicity of these compounds in the absence of light was also controlled. The results have shown that ethylenglycol esters can induce high levels of PpIX and are useful at concentrations below their cytotoxicity threshold. From the ALA-amino acid derivatives which were evaluated, the highest PpIX production was obtained using ALA derivatives of neutral amino acids, as compared to acidic or basic amino acids.
TL;DR: This work has shown that 5‐Aminolaevulinic acid (ALA) is a photosensitizer precursor that is transformed by cells into protoporphyrin IX (PpIX), which can in turn be activated by red light.
Abstract: Background Photodynamic therapy (PDT) is a new modality involving the administration of a photosensitizer, or photosensitizer precursor, followed by its activation with light to generate a therapeutic effect. 5-Aminolaevulinic acid (ALA) is a photosensitizer precursor that is transformed by cells into protoporphyrin IX (PpIX), which can in turn be activated by red light.
Objectives To investigate the effect of PDT in alopecia areata (AA).
Methods In six patients with extensive AA, topical ALA lotion at 5%, 10% and 20% as well as the vehicle lotion alone were applied separately to different scalp areas, followed 3 h later by exposure to red light at each treatment session.
Results No significant hair growth was observed after 20 twice-weekly treatment sessions. A significant increase in erythema and pigmentation was observed for the three concentrations of ALA lotion vs. the vehicle, implying that a phototoxic PDT effect was achieved in the skin. In vivo fluorescence spectroscopy in one patient showed an increase in red PpIX fluorescence 3 h after ALA application followed by a decrease after light exposure. On fluorescence microscopy, bright red fluorescence was present in the epidermis and sebaceous glands, but not in the inflammatory infiltrate surrounding the hair follicle following ALA application.
Conclusions PDT was ineffective in the treatment of AA.
TL;DR: ALA-n-hexylester appears to have slightly more favorable characteristics for PDT than ALA or ALA- n-butylester, although both ALA esters led to a more homogeneous PpIX localization.
Abstract: Our novel approach was to compare the pharmacokinetics of 5-aminolevulinic acid (ALA), ALA-n-butyl and ALA-n-hexylester induced protoporphyrin IX (PpIX), together with the phototoxicity after photodynamic therapy (PDT) in human skin in vivo, using iontophoresis as a dose-control system. A series of four increasing doses of each compound was iontophoresed into healthy skin of 10 volunteers. The kinetics of PpIX metabolism (n = 4) and the response to PDT (n = 6) performed 5 h after iontophoresis, were assessed by surface PpIX fluorescence and post-irradiation erythema. Whilst ALA-induced PpIX peaked at 7.5 h, highest PpIX fluorescence induced by ALA-n-hexylester was observed at 3-6 h and no clear peak was seen with ALA-n-butylester. With ALA-n-hexylester, more PpIX was formed after 3 (P < 0.05) and 4.5 h, than with ALA or ALA-n-butylester. All compounds showed a linear correlation between logarithm of dose and PpIX fluorescence/phototoxicity at 5 h, with R-values ranging from 0.87 to 1. In addition, the ALA-n-hexylester showed the tendency to cause greater erythema than ALA and ALA-n-butylester. Fluorescence microscopy (n = 2) showed similar PpIX distributions and penetration depths for the three drugs, although both ALA esters led to a more homogeneous PpIX localization. Hence, ALA-n-hexylester appears to have slightly more favorable characteristics for PDT than ALA or ALA-n-butylester.
TL;DR: Findings reinforce the importance of the vehicle in topical ALA‐based PDT, and explain the mechanism of action of DMSO in enhancing protoporphyrin IX biosynthesis in superficial lesions.
Abstract: Background The optimal vehicle to ensure adequate penetration of 5-aminolaevulinic acid (ALA) for its use in photodynamic therapy (PDT) of skin lesions has not been determined. Objectives We aimed to study the effects of ALA in various vehicle formulations [saline lotion with and without dimethylsulphoxide (DMSO), cream, liposomes and vaseline] after topical application in a murine subcutaneous adenocarcinoma model. Methods The effect of DMSO on porphyrin synthesis and ALA penetration through the skin was studied by measuring the uptake of 14 C label from ALA, ALA and porphobilinogen accumulation, and some haem enzyme activities. The tissue distribution and kinetics of porphyrin synthesis after topical application of ALA entrapped in large multilamellar liposomes was also determined. Results ALA in saline lotion, alone or with 10% DMSO, proved to be the most efficient vehicle for tumour porphyrin accumulation (mean ± SD 1.75 ± 0.25 and 2.09 ± 0.39 μg g -1 . respectively), whereas cream and liposomes induced lower levels and identical porphyrin accumulation (0.60 μg g -1 ). Using ALA + DMSO saline lotion, a higher porphyrin accumulation was found in skin overlying the tumour tissue and in the first 2 mm of tumour, probably due to increased ALA penetration, or greater interconversion to porphyrins, or greater retention of ALA and/or porphyrins. Conclusions These findings reinforce the importance of the vehicle in topical ALA-based PDT, and explain the mechanism of action of DMSO in enhancing protoporphyrin IX biosynthesis in superficial lesions.
TL;DR: Resonance Raman and UV-vis absorption spectroscopies of wild-type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into porphyrin, suggesting that distortion occurs in murineFerroChelatase for some porphirins, even without metal binding, which is apparently required for the yeast ferroCHLase.
Abstract: Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe2+ chelation into protoporphyrin IX. Resonance Raman and UV−vis absorption spectroscopies of wild-type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into porphyrin. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. On the basis of changes in the UV−vis absorption spectrum, addition of free-base or metalated porphyrins to wild-type ferrochelatase and H207N variant yields a 1:1 complex, most likely a monomeric protein-bound species at the active site. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is sub...
TL;DR: The available crystallographic data suggested that the distinct heme environments may contribute to the different inhibition mechanisms of HRP and CcO by cyanyl radical.
Abstract: Previous studies established that the cyanyl radical ((*)CN), detected as 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(*)CN by the electron spin resonance (ESR) spin-trapping technique, can be generated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H(2)O(2)) and by mitochondrial cytochrome c oxidase (CcO) in the absence of H(2)O(2). To investigate the mechanism of inhibition by cyanyl radical, we isolated and characterized the iron protoporphyrin IX and heme a from the reactions of CN(-) with HRP and CcO, respectively. The purified heme from the reaction mixture of HRP/H(2)O(2)/KCN was unambiguously identified as cyanoheme by the observation of the protonated molecule, (M + H)(+), of m/z = 642.9 in the matrix-assisted laser desorption/ionization (MALDI) mass spectrum. The proton NMR spectrum of the bipyridyl ferrous cyanoheme complex revealed that one of the four meso protons was missing and had been replaced with a cyanyl group, indicating that the single, heme-derived product was meso-cyanoheme. The holoenzyme of HRP from the reconstitution of meso-cyanoheme with the apoenzyme of HRP (apoHRP) showed no detectable catalytic activity. The Soret peak of cyanoheme-reconstituted apoHRP was shifted to 411 nm from the 403 nm peak of native HRP. In contrast, the heme a isolated from partially or fully inhibited CcO did not show any change in the structure of the protoporphyrin IX as indicated by its MALDI mass spectrum, which showed an (M + H)(+) of m/z = 853.6, and by its pyridine hemochromogen spectrum. However, a protein-centered radical on the CcO can be detected in the reaction of CcO with cyanide and was identified as the thiyl radical(s) based on inhibition of its formation by N-ethylmaleimide pretreatment, suggesting that the protein matrix rather than protoporphyrin IX was attacked by the cyanyl radical. In addition to the difference in heme structures between HRP and CcO, the available crystallographic data also suggested that the distinct heme environments may contribute to the different inhibition mechanisms of HRP and CcO by cyanyl radical.
TL;DR: The in vivo fluorescence kinetics showed that ALAPE induced more PpIX in visible lesions and altered skin of the UVB-exposed mouse skin, but not in the normal mouse skin; and higher ALAPE-induced Ppix levels were measured in the stratum corneum, butNot in the dysplastic layer of the epidermis.
Abstract: In order to improve the efficacy of 5-aminolevulinic acid-based (ALA) photodynamic therapy (PDT), different ALA derivatives are presently being investigated. ALA esters are more lipophilic and therefore may have better skin penetration properties than ALA, possibly resulting in enhanced protoporphyrin IX (PpIX) production. In previous studies it was shown that ALA pentyl ester (ALAPE) does considerably enhance the PpIX production in cells in vitro compared with ALA. We investigated the in vivo PpIX fluorescence kinetics after application of ALA and ALAPE to hairless mice with and without UVB-induced early skin cancer. ALA and ALAPE (20% wt/wt) were applied topically to the mouse skin and after 30 min, the solvent was wiped off and PpIX fluorescence was followed in time with in vivo fluorescence spectroscopy and imaging. At 6 and 12 h after the 30 min application, skin samples of visible lesions and adjacent altered skin (UVB-exposed mouse skin) and normal mouse skin were collected for fluorescence microscopy. From each sample, frozen sections were made and phase contrast images and fluorescence images were recorded. The in vivo fluorescence kinetics showed that ALAPE induced more PpIX in visible lesions and altered skin of the UVB-exposed mouse skin, but not in the normal mouse skin. In the microscopic fluorescence images, higher ALAPE-induced PpIX levels were measured in the stratum corneum, but not in the dysplastic layer of the epidermis. In deeper layers of the skin, PpIX levels were the same after ALA and ALAPE application. In conclusion, ALAPE does induce higher PpIX fluorescence levels in vivo in our early skin cancer model, but these higher PpIX levels are not located in the dysplastic layer of the epidermis.
TL;DR: The results indicate that the combination of 5-ALA mediated PDT and γ-irradiation results in a level of cytotoxicity which is additive and not synergistic.
Abstract: Photodynamic therapy (PDT) is a promising treatment modality for head and neck, and other tumours, using drugs activated by light. A second generation drug, 5-aminolaevulinic acid (5-ALA), is a precursor of the active photosensitizer protoporphyrin IX (PpIX) and has fewer side-effects and much more transient phototoxicity than previous photosensitizers. We have investigated the effect of 5-ALA mediated PDT in combination with γ-irradiation on the colony forming ability of several human head and neck tumour cell lines. The effect of treatments on the DNA cell cycle kinetics was also investigated. Our results indicate that the combination of 5-ALA mediated PDT and γ-irradiation results in a level of cytotoxicity which is additive and not synergistic. 5-ALA mediated PDT had no discernible effect on DNA cell cycle distributions. γ-irradiation-induced cell cycle arrest in G2 did not enhance the phototoxicity of 5-ALA. © 2000 Cancer Research Campaign
TL;DR: In preliminary clinical tests, a clear demarcation between neoplastic/cancerous lesions and adjacent normal tissue was demonstrated and laboratory tests of fluorescence ratio imaging showed good contrast enhancement between control tissues and tissue phantoms and those containing porphyrin photosensitisers.
Abstract: Topical or systemic administration of 5-aminolaevulinic acid results in biosynthesis of the photosensitiser protoporphyrin IX (PpIX) with some selectivity for malignant lesions. Excitation near 400 nm excites both intrinsic green tissue autofluorescence and red fluorescence from PpIX which may be exploited for the optical diagnosis of malignant and premalignant disease. In this work the utility of a cooled 12-bit single chip charge-coupled device (CCD) colour camera was investigated for photodiagnostic fluorescence ratio imaging. The red to green fluorescence intensity ratios were calculated for each pixel in real-time and fluorescence ratio images were displayed typically at a rate of 2 frames/s. Laboratory tests of fluorescence ratio imaging showed good contrast enhancement between control tissues and tissue phantoms and those containing porphyrin photosensitisers. In preliminary clinical tests, a clear demarcation between neoplastic/cancerous lesions and adjacent normal tissue was demonstrated. The extent of PpIX photobleaching during photodynamic therapy was also investigated using fluorescence ratio imaging.
TL;DR: Fluorescence detection of premalignancies after exogenous application of 5-aminolevulinic acid, which is converted to protoporphyrin IX and accumulates selectively in tumors, is an interesting approach.
TL;DR: In this paper, the central metal ion plays a significant role in the efficacy of metalloprotoporphyrins to inhibit malarial hemozoin formation, a critical detoxification biopolymer of malarial parasites.
TL;DR: Observations suggest that a marked heterogeneity of ALA uptake and/or PplX synthesis exists in a given human cancer cell population particularly after systemic administration of Delta-aminolevulinic acid (ALA)-PDT.
Abstract: Delta-aminolevulinic acid (ALA)-PDT efficacy is particularly dependent on the quality of protoporphyrin IX (PpIX)-induced synthesis. The purpose of this study was to determine the ability of cells from two human cancer types to synthesise PpIX after ALA administration. Biopsies of glioma cells have been obtained from patients with glioblastomas that have or have not been given ALA IV (ex vivo incubation). Peripheral blood lymphocytes, obtained from leukemic patients, have also been ALA-incubated in vitro. In glioma cells, fluorescence heterogeneity was extensive either in ALA infused patients or in ex vivo ALA incubated cells. Mean intensities after 3 h were 110 cts (range 0-340) and 1000 cts (range 0-3600). Similar results were found in leukemic lymphocytes where cell fluorescence varied from 0 to 480 cts with a percentage of fluorescent cells varying with time and from one patient to another. Furthermore, PpIX was not detectable in two patients with CLL. These observations suggest that a marked heterogeneity of ALA uptake and/or PpIX synthesis exists in a given human cancer cell population particularly after systemic administration. Improvements for ALA transformation into PpIX are strongly recommended to ensure the efficacy of ALA/PpIX-PDT.
TL;DR: expression of the green fluorescent protein (EGFP) transgenes driven by various ferrochelatase promoter fragments into a single locus in mouse embryonic stem cells was introduced to investigate the role of these elements during erythropoiesis and the effect of an A-to-G polymorphism identified in the promoters of patients with protoporphyria.