Identification of mutations in the uroporphyrinogen III cosynthase gene in German patients with congenital erythropoietic porphyria.

The porphyrias are heterogeneous disorders arising from predominantly inherited catalytic deficiencies of specific enzymes along the heme biosynthetic pathway. Congenital erythropoietic porphyria is a very rare disease that is inherited as an autosomal recessive trait and results from a profound deficiency of uroporphyrinogen III cosynthase, the fourth enzyme in heme biosynthesis. The degree of severity of clinical symptoms mainly depends on the amount of residual uroporphyrinogen III cosynthase activity. In this study, we sought to characterize the molecular basis of congenital erythropoietic porphyria in Germany by studying four patients with congenital erythropoietic porphyria and their families. Using PCR-based techniques, we identified four different mutations: C73R, a well-known hotspot mutation, the promoter mutation -86A that was also described previously, and two novel missense mutations, designated G236V and L237P, the latter one encountered in the homozygous state in one of the patients. Our data from the German population further emphasize the molecular heterogeneity of congenital erythropoietic porphyria as well as the advantages of molecular genetic techniques as a diagnostic tool and for the detection of clinically asymptomatic heterozygous mutation carriers within families.

Photosensitization of uroporphyrin augments the ultraviolet A-induced synthesis of matrix metalloproteinases in human dermal fibroblasts.

Porphyria cutanea tarda is characterized by severe connective tissue damage in sun-exposed skin. The regulated synthesis and degradation of the extracellular matrix by various matrix metalloproteinases (MMPs) determine its amount and composition within the skin. In this study, we therefore asked whether long-wave ultraviolet irradiation (340-450 nm) in conjunction with uroporphyrin I could modulate the synthesis of MMPs with substrate specificities for dermal (collagens I, III, V; proteoglycans) and basement membrane components (collagens IV, VII; fibronectin; laminin) and whether synthesis of the counteracting tissue inhibitor of metalloproteinases is also affected. After irradiation of uroporphyrin-pretreated fibroblasts, specific mRNAs of MMP-1 and MMP-3 increased concomitantly up to 2.7-fold compared with ultraviolet-irradiated cells and up to 10-fold compared with mock-irradiated or uroporphyrin I-treated controls. In contrast, mRNA levels of tissue inhibitor of metalloproteinases remained unaltered. Similar results were obtained by immunoprecipitation. Gelatin and casein zymography revealed increased proteolytic activity of MMP-2 and MMP-3 in blister fluids of patients with porphyria cutanea tarda, indicating that similar events may occur in vivo. Using deuterium oxide as enhancer and sodium azide as quencher of singlet oxygen, we could increase or reduce MMP synthesis, suggesting that singlet oxygen is the major intermediate in the upregulation of MMPs after irradiation of uroporphyrin-pretreated fibroblasts. Taken together, our results show that ultraviolet irradiation alone, and to a greater extent in conjunction with uroporphyrin I, results in an unbalanced synthesis of MMPs that may contribute to the destruction of the dermis and basement membrane, leading to blistering and accelerated photoaging in porphyria cutanea tarda patients.

UVA-induced autocrine stimulation of fibroblast-derived-collagenase by IL-6: a possible mechanism in dermal photodamage?

Like other cytokines, IL-6 has been reported to stimulate collagenase. In this study we were interested in whether IL-6 is involved in the ultraviolet (UV) mediated up-regulation of fibroblast-derived collagenase. Confluent fibroblast monolayers were irradiated under standardized conditions. Following UVA irradiation the bioactivity of IL-6 increased up to fiftyfold in the supernatants of irradiated compared to mock-irradiated fibroblasts. As determined by Northern blot analysis this was also reflected on the pre-translational level by a tenfold increase of IL-6-specific mRNA following UVA irradiation. Induction of IL-6-specific mRNA was maximal at 6 h post-irradiation, thus clearly preceding the maximal induction of collagenase mRNA at 24 h post-irradiation. To elucidate the regulatory role of IL-6 in the UVA induction of fibroblast-derived collagenase, monospecific polyclonal neutralizing antibodies directed against recombinant human IL-6 and antisense oligonucleotides specifically inhibiting the translation of IL-6 mRNA were used at various concentrations. The amount of UVA-induced collagenase mRNA was reduced in a dose-dependent manner when antibodies or specific antisense oligonucleotides were present during and after irradiation. Taken together our data provide first evidence that UVA enhances IL-6 synthesis and secretion in fibroblasts. IL-6 induces via an autocrine mechanism collagenase and may thus contribute to the actinic damage of the dermis.

Effect of hexachlorobenzene on the activities of hepatic alcohol metabolizing enzymes.

To study the effect of experimental hepatic porphyria on the activities of hepatic alcohol metabolizing enzymes, female rats received a chow diet containing 0.05% hexachlorobenzene (HCB). After long-term HCB treatment for 60 days hepatic porphyria developed as evidenced by increased hepatic delta-aminolevulinic acid synthase activity and enhanced urinary excretion of delta-aminolevulinic acid, porphobilinogen and total porphyrins. Concomitantly, the activities of the hepatic microsomal ethanol oxidizing system (MEOS) were strikingly augmented by 213% (P less than 0.05) and 177% (P less than 0.01) when expressed per g of liver wet weight or per 100 g of body weight, respectively, whereas hepatic alcohol dehydrogenase activities remained virtually unchanged. Moreover, hepatic catalase showed only a trend for a slightly lower enzymic activity under these experimental conditions. The present data therefore show that experimental hepatic porphyria is associated with alterations of hepatic MEOS activities, which in turn may be a factor for the manifestation of human hepatic porphyrias in the course of alcohol consumption.

Aminolevulinic acid: pharmacological profile and clinical indication.

The role of aminolevulinic acid hydrochloride (ALA) in photodynamic therapy (PDT) of in situ neoplasias and tumours of epithelial tumours is steadily increasing and it has been shown to be the drug with most clinical use in PDT. In dermatology, topical PDT with ALA is already postulated to be the treatment of choice for actinic keratoses and superficial basal cell carcinomas. In gastroenterology, pulmonology, uro- and nephrology, neurology and gynaecology ALA has an important role as a photosensitiser not only in the diagnosis of neoplastic tissue but as therapy; first experiences have been made with PDT in these organs. Besides the therapeutic efficacy of this technique, the fluorescence of ALA-induced porphyrins can be effectively used to detect and delineate epithelial and endothelial neoplasms. In dermatology, other indications for ALA-treatment are non-tumoural applications, especially psoriasis, viral-induced diseases, or acne vulgaris. ALA is an effective compound in the diagnosis or therapy of various epithelial and endothelial neoplastic lesions.

Technical and economic feasibility of reusing disposable perfusion cannulas.

OBJECTIVE(S): The reuse of disposable devices is a potential source of significant cost savings to hospitals. Venous and arterial perfusion cannulas under new and reused conditions were selected to identify the clinical, safety, technical, logistic, and economic issues that must be addressed to realize these savings. METHODS: Single- and dual-stage venous and arterial cannulas from two manufacturers were tested when new, after initial clinical use, and after a single clinical use plus up to nine simulated reuses. Reuse was simulated by end-to-end bending, coupling and uncoupling of the connectors, and by two 1-hour soaks in plasma at 4 degrees and 40 degrees C, respectively. Cannulas were decontaminated and then processed by a peracetic acid-based liquid chemical sterilization system after each use/reuse. Sterilization was validated by eliminating Bacillus subtilis spores from the cannulas on each of five consecutive cycles. Cannulas were tested for physical changes, functional integrity, biocompatibility, and in vivo performance in sheep. A cost analysis was also performed. RESULTS: Sterilization was successfully achieved. Mechanical changes were less than 20% on all variables studied and were undetectable by experienced cardiac surgeons in selective evaluation. No clinically important differences were found between new and reused cannulas, even after nine simulated reuses. Reusing cannulas four times would reduce the cost per procedure from $53 to $19 (64%). CONCLUSIONS: Preliminary data suggest that the perfusion cannulas tested can be safely and efficaciously used five times. Limited reuse of these disposable cannulas is technically feasible and cost-effective. Cannula reuse would result in a small incremental savings; however, with more expensive devices and higher-volume sterilization procedures, the savings could be considerably greater. This program provides a model for evaluation of other single-use medical devices for reuse.

Ex vivo application of delta-aminolevulinic acid induces high and specific porphyrin levels in human skin tumors: possible basis for selective photodynamic therapy.

In photodynamic therapy with topically applied delta-aminolevulinic acid porphyrins are acting as photosensitizers. The profile of porphyrin metabolites in normal or in neoplastic skin after administration of delta-aminolevulinic acid has not been determined in detail yet. Thus, to study porphyrin biosynthesis in human skin an organ culture model was developed. Explant pieces of normal skin, keratoacanthoma, and basal cell carcinoma were incubated with 1 mM delta-aminolevulinic acid for 36 h. Levels of delta-aminolevulinic acid, porphyrins and porphyrin metabolites were measured in tissues and supernatants. After incubation with delta-aminolevulinic acid, higher porphyrin levels were demonstrated in tumors as compared to normal skin. In supernatants, most of formed porphyrins, preferentially highly carboxylated porphyrin metabolites, were measured. The pattern of synthesized porphyrins differed between normal and neoplastic skin explants. In tissues of basal cell carcinomas protoporphyrin was preferentially shown and tissues of keratoacanthomas were characterized by a predominance of coproporphyrin as compared to normal skin. The results show that explant cultures offer an easy approach to examine the porphyrin biosynthesis of various tissues. The tumor-specific delta-aminolevulinic acid metabolism indicates additional porphyrin metabolites such as coproporphyrin apart from protoporphyrin as effective photosensitizers and may offer a novel approach to tumor-selective photodynamic damage.

Influence of chloroquine on the porphyrin metabolism.

Rats were treated with the well-known porphyrogen hexachlorobenzene (HCB) to induce experimental porphyria. At the same time another group of rats was treated with chloroquine in addition to HCB. The HCB-induced increase of the urinary excretion of porphyrin precursors could thereby be reduced to normal levels and the porphyrin excretion rates were decreased significantly in comparison to those of the other group. The delta-aminolevulinate synthase in the liver of the animals was slightly increased by exclusive treatment with chloroquine, which in the HCB-treated rats chloroquine led to a dramatic decrease in the key enzyme of the porphyrin (heme)-biosynthesis. The influence of chloroquine on the HCB-induced increase of the cytochrome P-450 content and the dependent enzymatic activities were different. The 7-ethoxycumarin deethylase and the arylhydrocarbon hydroxylase activities were not influenced, whereas the increased aminopyrine-N-demethylase activity was reduced to nearly normal levels. Our findings indicate that chloroquine acts by reduction of the delta-aminolevulinate synthase activity, probably by influencing the regulation of the key enzyme of the heme biosynthesis, which is enhanced in human porphyria cutanea tarda, as well as in the HCB-induced porphyria of the rats.

Influence of oral isotretinoin on hepatic and cutaneous P-450-dependent isozyme activities.

Oral administration of isotretinoin (13-cis-retinoic acid) (6 mg/kg per day), 0.05% hexachlorobenzene (HCB) or both drugs simultaneously for 10 days to female Wistar rats caused a statistically significant induction of aminopyrine-N-demethylase (ADM), 7-ethoxyresorufin-O-deethylase (7-ERO-D) and erythromycin-N-demethylase (EMDM) in the liver microsomes. Oral administration of isotretinoin alone or together with HCB induced a marked induction of 7-ERO-D and EMDM in the skin. Administration of isotretinoin alone for 60 days resulted in the induction of EMDM in the liver microsomes, and in combination with HCB caused a statistically significant induction of all hepatic isozymes. HCB alone caused a marked induction of only 7-ERO-D in the skin. These results clearly show that oral isotretinoin is capable of inducing hepatic and cutaneous microsomal P-450-dependent catalytic activities. It remains to be elucidated whether the induction of these enzymes is of importance for the therapeutic action of isotretinoin.

[Hexachlorbenzene (HCB) induced porphyria in rats. Influence of HCB-metabolites on the biosynthesis of heme (author's transl)]. / Hexachlorbenzol (HCB)-bedingte porphyrie der Ratte. Einfluss von HCB-Metaboliten auf die Haembiosynthese.

Female adult Wistar rats were fed with a diet containing 0.05% hexachlorobenzene (HCB) or its metabolites, pentachlorobenzene (PCB) and pentachlorphenole (PCP). These chlorinated aromatic hydrocarbons produced an increase in the liver cytochrome P-450 content in about the same degree, however, only the application of HCB showed an extremely high rise in the P-450 enzymatic activity expressed in terms of the O-dealkylation of 7-Ethoxycoumarine. No alteration was observed in the urinary porphyrin excretion in the PCB and PCP treated animals, whereas 60 days after the beginning of the HCB application a high level of porphyrins could be detected in the urine of the animals. It seems unlikely therefore that the HCB metabolites (PCB and PCP) are porphyrogenic agents. In addition, although induction of the liver cytochrome P-450 system was observed after PCP pretreatment of the rats over a period of 40 days, the consequent application of HCB did not influence the establishment of the experimental porphyria.

Effects of oral thalidomide on rat liver and skin microsomal P450 isozyme activities and on urinary porphyrin excretion: interaction with oral hexachlorobenzene.

Adult female Wistar rats (n = 48) divided into four groups of 12 were treated orally with 3 mg/kg per day thalidomide, a 0.05% hexachlorobenzene (HCB)--containing diet, with both drugs together and with the vehicles (controls) over periods of 10 and 60 days. The protein and P450 contents and the activities of aminopyrine-N-demethylase (ADM) and 7-ethoxyresorufin-O-deethylase (7-ERO-D) were determined in the liver microsomes. The activity of 7-ERO-D was also determined in the skin microsomes and total porphyrins were measured in the urine of the animals. Thalidomide increased the hepatic P450 content, caused distinct changes in the activities of the hepatic and cutaneous microsomal isozymes, modified their induction by HCB and inhibited the porphyrogenic activity of HCB. These findings indicate an interaction between thalidomide and HCB with regard to their effects on the P450 isozymes and the metabolism of porphyrins.

[Influence of chloroquine on the hexachlorobenzene-induced porphyria. Investigations in the skin, liver, and urine (author's transl)]. / Einfluss von Chloroquin auf die Hexachlorbenzol-induzierte Porphyrie. Untersuchungen in Haut, Leber und Urin.

Rats were fed with a diet containing hexachlorobenzene (HCB) for about 60 days. At this time the porphyria was manifst as shown by significantly elevated porphyrins. Thereafter, chloroquine (CQ) was additionally given over a period of at lest 6 weeks. At the end of the experiment the urinary porphyrin excretion and the porphyrin content in lijver and skin were diminished in HCB-CQ-treated animals by about 50% compared to the HCB controls. The relative porphyrin distribution pattern was not influenced by CQ. In a further investigation the prophylactic effect of CQ could be demonstrated. Rats given CQ simultaneously from the beginning of the HCB feeding showed a significantly delayed onset of porphyria. It is concluded from our results that CQ does not only form complexes with porphyrins during the treatment of th HCB porphyria. We rather assume an effect of CQ on the metabolism of iron.

Influence of topical photodynamic therapy with 5-aminolevulinic acid on porphyrin metabolism.

Photodynamic therapy (PDT) with topically applied 5-aminolaevulinic acid (5-ALA) is increasingly used for treating tumours. The efficacy of topical PDT is limited to superficial and initial tumours. The topically applied doses of 5-ALA vary from 0.02 to 7.0 g per session according to the type of lesion. There are no studies on the influence of topically applied 5-ALA on the systemic accumulation of porphyrins or porphyrin precursors. A group of 20 patients with actinic keratoses (AK) and basal cell carcinomas (BCC) were treated by topical PDT with 5-ALA. Prior to and 6 and 24 h after PDT, 5-ALA and total porphyrin concentrations were determined in red blood cells and plasma, respectively. In addition, before and after 5-ALA treatment, 24-h urine samples were collected and porphyrins and porphyrin precursors were measured. There was no significant alteration in porphyrin metabolism. In some patients, a slight but insignificant increase in erythrocyte and plasma porphyrins was found 6 h after 5-ALA PDT. This investigation confirms clearly the safety of this treatment modality and demonstrates that 5-ALA application (up to 7 g) in the course of PDT has no influence on the concentrations of porphyrins and porphyrin precursors measured in various compartments.

UVA irradiation induces collagenase in human dermal fibroblasts in vitro and in vivo.

We report the effect of UVA irradiation on collagen metabolism of fibroblasts, including both synthesis of the collagen degrading enzyme collagenase and de novo synthesis of type I collagen as the major structural component of the dermis. For this purpose confluent fibroblast monolayers were irradiated under standardized conditions (5, 15, 35, 60 J/cm2 using UVASUN 3000, Mutzhas, Munich, FRG, and UV source Sellas sunlight type 2.001, Sellas, Gevelsberg, FRG). Subsequently, total RNA was isolated and subjected to dot blot and northern blot analysis using oligolabelled cDNA clones for human type I collagen, collagenase and beta-actin. Collagen type I and beta-actin mRNA levels remained unaltered following irradiation, suggesting that the synthetic pathway of collagen metabolism at the pretranslational level is not affected by short-term UVA irradiation. However, collagenase mRNA was found to be dose-dependently induced in fibroblasts after irradiation, thus probably contributing to the actinic damage to the dermis. These in vitro data were confirmed in vivo using in situ hybridization on frozen sections of biopsy material obtained from UVA irradiated patients.

Influence of UVA and UVB irradiation on hepatic and cutaneous P450 isoenzymes.

The influence of UVA and UVB irradiation of the skin for 1, 2 and 4 weeks on the activities of the hepatic and cutaneous P450 isoenzymes was investigated in female Wistar rats before and after systemic administration of hexachlorobenzene (HCB), a well-known porphyrogenic agent, which additionally induces P450 1A1 and P450 1A2 isoenzymes. UVA and UVB irradiation of the skin of controls and HCB-treated animals did not influence porphyrin metabolism. In the nonporphyric rats hepatic EROD (P450 1A1) activity was induced by UVB, but the activity of ADM (P450 2B) and EMDM (P450 3A) was either minimally or not affected. In the HCB-treated (porphyric) rats UVA and UVB irradiation resulted in a significant depression of HCB-induced EROD in the liver and in the skin. In both the nonporphyric and the porphyric rats UVA and UVB irradiation had no effect on hepatic ADM activity. In the liver of the nonporphyric animals EMDM activity remained unchanged after UVA and UVB irradiation, whereas in the HCB-treated animals the activity of this enzyme was increased. Finally, after UVA and UVB irradiation cutaneous EMDM activity was increased in the controls, whereas the HCB-induced increase of this enzyme in porphyric animals was decreased. In addition long-term (28 days) UVB irradiation decreased hepatic GSH content significantly in normal and porphyric rats. These experimental findings cannot be directly extrapolated to humans; however, they suggest that exposure of human skin to UV radiation may result in alterations in the activity of cutaneous hepatic and other extracutaneous P450 isoenzymes.

Beta-carotene serum levels in patients with erythropoietic protoporphyria on treatment with the synthetic all-trans isomer or a natural isomeric mixture of beta-carotene.

The all-trans-beta-carotene serum level of patients suffering from erythropoietic protoporphyria increases substantially during continuous treatment with beta-carotene (either with the synthetic all-trans compound or with beta-carotene from a natural source consisting of a cis-trans isomeric mixture). On continuous daily ingestion, the beta-carotene serum level rose from day 0 to day 30, and no further increase was observed between day 30 and day 150. Slightly lower beta-carotene steady state serum levels were observed with the natural isomeric mixture than with synthetic beta-carotene. Higher levels of 13-cis-beta-carotene, in some cases up to 10% of the total beta-carotene, were detected after ingestion of the synthetic compound. The level of 9-cis-beta-carotene was below or close to the limit of quantification in all samples, even when the isomeric mixture containing high amounts of 9-cis-beta-carotene was applied.

The 5-aminolevulinic acid-induced porphyrin biosynthesis in benign and malignant cells of the skin.

In fluorescence diagnosis and photodynamic therapy of neoplastic tissues 5-aminolevulinic acid is used to synthesize endogenous porphyrins as photosensitizers. The efficacy of neoplastic tissues to fluorescence diagnosis and photodynamic therapy is thought to be dependent on the total level of intralesional formed porphyrins. The available profiles of porphyrin metabolites in normal and in neoplastic cell lines after administration of 5-aminolevulinic acid vary considerably. Thus, this is the first in-vitro study which compares the porphyrin biosynthesis in normal skin cells (HaCaT, fibroblasts) with melanoma cells (Bro, SKMel-23, SKMel-28). After incubation with 1 mM 5-aminolevulinic acid, kinetics of porphyrin levels and metabolites were determined in the cells and the corresponding supernatants. Exogenous 5-aminolevulinic acid induced porphyrin formation in all cells with maximum values after an incubation period of 16-36 h. Increase of porphyrin levels varied from 10- to 80-fold (SKMel-28>HaCaT>fibroblasts>SKMel-23>>Bro) with minimum 1.5 times higher levels of porphyrins in the supernatants than in the cells. In cells and supernatants protoporphyrin and coproporphyrin were the predominantly formed porphyrin metabolites. Metastatic melanoma cells (SKMel-23, SKMel-28) accumulated much higher porphyrin levels than primary melanoma cells (Bro). In conclusion, by optimizing the treatment modalities, especially the light source, topical photodynamic therapy (PDT) could become a treatment alternative of melanoma metastases in progressive disease.

Formation of water-soluble porphyrins and protoporphyrin IX in 5-aminolevulinic-acid-incubated carcinoma cells.

The bioconversion of 5-aminolevulinic acid (ALA) into hydrophobic protoporphyrin IX and other water-soluble porphyrins was investigated in Ehrlich ascite carcinoma (EAC) cells and in a myeloma cell line. The effects of irradiation (514 nm), temperature, incubation time and added glucose on the relative porphyrin concentrations (protoporphyrin vs. water-soluble porphyrins) were examined. Variations in these parameters induced a change in the amount of water-soluble porphyrins relative to protoporphyrin IX. The main component of the hydrophilic porphyrins was found to be uroporphyrin (Up), with minor components of coproporphyrin (Cp) and other carboxyporphyrins. The enhanced production of water-soluble porphyrins appears to be associated with alterations in the activities of the various enzymes in the heme biosynthetic pathway, resulting, for example, in the reduction in the activity of mitochondrial enzymes.

Photodynamic therapy with 5-aminolaevulinic acid-induced porphyrins of an amelanotic melanoma in vivo.

Of particular interest for photodynamic therapy (PDT) are the endogenously formed and photodynamically active porphyrins produced following topical or systemic application of 65-aminolaevulinic acid (ALA), a haem precursor. Having determined the pharmacokinetics and wavelength dependence of PDT with ALA-induced porphyrins, we analysed the porphyrin metabolites in tumour and surrounding skin. The therapeutic efficacy of PDT using ALA-induced porphyrins was investigated. Amelanotic melanomas (A-Mel-3) were implanted subcutaneously in the back of Syrian golden hamsters (body weight (b.w.), 70-80 g). After 5-7 days, tumours with a volume of approximately 150 mm3 were used for PDT (n = 36). ALA (500 mg kg-1 b.w., pH 6.5) was injected intravenously 45, 90, 150 and 300 min before light irradiation (635 nm, 100 mW cm-2, 100 J cm-2). Tumours with light irradiation only served as controls. The tumour volume was measured after PDT for 28 days. The total porphyrin content was determined in the tumours, the surrounding skin and erythrocytes prior to and 45, 90, 180, 240, 300 and 480 min and 24 h following intravenous injection of ALA (500 mg kg-1 b.w.; n = 32). Porphyrin metabolites were separated by high pressure liquid chromatography (HPLC). Tumour growth was significantly delayed when PDT with ALA was performed 45, 90 or 150 min following intravenous administration. At that time, protoporphyrin (1.8 +/- 0.4 nmol g-1), coproporphyrin (2.2 +/- 0.5 nmol g-1) and uroporphyrin (1.7 +/- 1.4 nmol g-1) were the main metabolites in the tumour tissue. Erythrocytes also contained significant amounts of porphyrins (11.8 +/- 1.3 nmol g-1). The tumour and surrounding skin exhibited a different pattern of porphyrin metabolites. Unexpectedly, a single treatment of PDT with ALA-induced porphyrin resulted in only one complete remission out of six amelanotic melanomas when the final therapeutic outcome was assessed after 28 days. The therapeutic efficacy of PDT with ALA-induced porphyrins can be positively correlated with the fluorescence kinetics previously determined. The analysis of the porphyrin metabolites in amelanotic melanoma by HPLC indicates that the porphyrin accumulation is not due to a decreased activity of ferrochelatase. Moreover, the photodynamic effects may not be mediated solely by porphyrins localized in the tumour parenchyma, but also by significant amounts of porphyrins in the microvasculature. PDT with this endogenous photosensitizer failed to induce complete emission of the treated tumours despite irradiation at the time of maximum porphyrin concentration using the optimum therapeutic wavelength. Thus PDT with ALA-induced porphyrins is less effective in our model relative to that observed for the exogenous photosensitizer Photofrin or synthetic porphycenes after a single treatment.