Application Of 5-aminolevulinic Acid Research Articles

The Exogenously Applied 5-Aminolevulinic Acid (ALA) Mitigates Salt Stress in Tomato Plants

The Exogenously Applied 5-Aminolevulinic Acid (ALA) Mitigates Salt Stress in Tomato Plants

Noninvasive Optical Imaging of UV-Induced Squamous Cell Carcinoma in Murine Skin: Studies of Early Tumor Development and Vitamin D Enhancement of Protoporphyrin IX Production.

Better noninvasive techniques are needed to monitor protoporphyrin IX (PpIX) levels before and during photodynamic therapy (PDT) of squamous cell carcinoma (SCC) of the skin. Our aim was to evaluate (1) multispectral fluorescent imaging of ultraviolet light (UV)-induced cancer and precancer in a mouse model of SCC and (2) multispectral imaging and probe-based fluorescence detection as a tool to study vitamin D (VD) effects on aminolevulinic acid (ALA)-induced PpIX synthesis. Dorsal skin of hairless mice was imaged weekly during a 24-week UV carcinogenesis protocol. Hot spots of PpIX fluorescence were detectable by multispectral imaging beginning at 14 weeks of UV exposure. Many hot spots disappeared after cessation of UV at week 20, but others persisted or became visible after week 20, and corresponded to tumors that eventually became visible by eye. In SCC-bearing mice pretreated with topical VD before ALA application, our optical techniques confirmed that VD preconditioning induces a tumor-selective increase in PpIX levels. Fluorescence-based optical imaging of PpIX is a promising tool for detecting early SCC lesions of the skin. Pretreatment with VD can increase the ability to detect early tumors, providing a potential new way to improve efficacy of ALA-PDT.

Intraoperative photodynamic diagnosis of lymph node metastasis in esophageal cancer patients using 5-aminolevulinic acid.

Lymph node metastasis is the strongest prognostic factor in esophageal cancer patients who have undergone esophagectomy. The accurate diagnosis of lymph node metastasis is important, but the pre-operative diagnostic accuracy is poor. The intraoperative diagnosis based on histopathological examination of frozen tissue specimens is complicated and time-consuming. Therefore, the establishment of a simple and rapid intraoperative diagnostic method is essential. Exogenous application of 5-aminolevulinic acid (ALA) causes a selective accumulation of protoporphyrin IX, which is a fluorescent substrate, in cancer cells. The present study evaluated the feasibility of photodynamic diagnosis using ALA (ALA-PDD) for lymph node metastasis in esophageal cancer. A total of 292 lymph nodes were analyzed from 8 esophageal squamous cell cancer patients treated with esophagectomy. The patients were administered ALA orally prior to surgery. Excised lymph nodes were cut in half and examined by spectrometer. The diagnostic results of ALA-PDD were compared to those of the histopathological examination. Among the 292 lymph nodes, 19 nodes (6.5%) were histologically metastatic and 21 nodes (7.2%) were PDD-positive. The sensitivity and specificity of ALA-PDD were 84.2% (16/19) and 98.2% (268/273), respectively. The area of cancer nests of the PDD-negative lymph nodes was <2 mm2. Metastatic lymph nodes, including cancer nests >4 mm2, were correctly diagnosed by ALA-PDD. In conclusion, this study demonstrated that ALA-PDD of lymph node metastasis in patients with esophageal cancer is feasible. Further investigation would make this method a simple and rapid intraoperative diagnostic tool.

Pale-green phenotype of atl31atl6 double mutant leaves is caused by disruption of 5-aminolevulinic acid biosynthesis in Arabidopsis thaliana.

Arabidopsis ubiquitin ligases ATL31 and homologue ATL6 control the carbon/nitrogen nutrient and pathogen responses. A mutant with the loss-of-function of both atl31 and atl6 developed light intensity-dependent pale-green true leaves, whereas the single knockout mutants did not. Plastid ultrastructure and Blue Native-PAGE analyses revealed that pale-green leaves contain abnormal plastid structure with highly reduced levels of thylakoid proteins. In contrast, the pale-green leaves of the atl31/atl6 mutant showed normal Fv/Fm. In the pale-green leaves of the atl31/atl6, the expression of HEMA1, which encodes the key enzyme for 5-aminolevulinic acid synthesis, the rate-limiting step in chlorophyll biosynthesis, was markedly down-regulated. The expression of key transcription factor GLK1, which directly promotes HEMA1 transcription, was also significantly decreased in atl31/atl6 mutant. Finally, application of 5-aminolevulinic acid to the atl31/atl6 mutants resulted in recovery to a green phenotype. Taken together, these findings indicate that the 5-aminolevulinic acid biosynthesis step was inhibited through the down-regulation of chlorophyll biosynthesis-related genes in the pale-green leaves of atl31/atl6 mutant.

Involvement of nitrate reductase in the ameliorating effect of 5-aminolevulinic acid on NaCl-stressed barley seedlings

Exogenous application of 5-aminolevulinic acid (ALA) is widely used to decrease the effect of different abiotic stresses in various crops while the nitrogen metabolism is a possible target of ALA in plants. We investigated the effect of exogenous ALA on growth, activity of nitrate reductase (NR; EC 1.6.6.1), synthesis of proline and ROS generation in seedlings of the salt-sensitive barley cultivar (Hordeum vulgare L. cv. Gonar). Obtained data indicated that the ALA (20, 40 and 80 mg L−1) increased elongation and leaf blade expansion of barley seedlings exposed to 150 mM NaCl as compared to control plants, which were not treated by ALA. NR gene (Nar1) expression, NR protein content and enzymatic activities increased in ALA-treated plants. ALA also induced accumulation of free proline and decreased generation of superoxide anion radicals. Overall, this study demonstrates that ALA effects on salt-stressed barley involves changes of NR activities both at transcriptional and translational levels and leads to complex physiological modifications, such as improvement of growth and antiradical defense.

Induction of protoporphyrin IX in patient-derived synoviocytes, cartilage explants and chondrons after application of 5-aminolevulinic acid or its methyl ester

ObjectiveTo compare the accumulation of protoporphyrin IX between synoviocytes of patients with rheumatoid arthritis (RA) or osteoarthritis (OA) and cartilage explants (CE) as well as chondrons of patients with OA after the application of 5-aminolevulinic acid (ALA) or its methyl ester (ALA-Me). Materials and methodsSamples of synovial and cartilage tissues were obtained from joint replacement surgeries. The accumulation of PpIX was determined by measuring fluorescence spectra from 2×105 synoviocytes or chondrons suspended in a glass tube or directly from CE surface after 2, 4, 8 and 24h of incubation with ALA or ALA-Me. ResultsNo differences were found between the average fluorescence intensity values of PpIX in synoviocytes of patients with RA and OA. These values were non-significantly higher after incubation with ALA in comparison with ALA-Me at almost all time points. The average fluorescence intensity of PpIX in CE and chondrons was about ten times lower than in synoviocytes. The presence of preparation of hyaluronic acid (HA) significantly enhanced PpIX induction in chondrons versus treatment only with ALA. ConclusionsA potential for the selective synovial sensitization with endogenous PpIX in comparison with cartilage tissue has been demonstrated in vitro after application of ALA or ALA-Me.

Response of Nitrate Reductase to Exogenous Application of 5-Aminolevulinic Acid in Barley Plants

Information on mechanisms and the pathway for plant nitrogen assimilation by 5-aminolevulinic acid (ALA) is still limited. In addition, the molecular mechanism of nitrate reductase (NR) regulation in response to ALA treatment in plants has not been fully elucidated. In this study, we investigate the effect of different concentrations of exogenous ALA on expression, protein content, and enzyme activity of NR in 7-day-old barley (Hordeum vulgare L.) seedlings grown in the presence of its substrate, KNO3. Our data indicate that the nitrate-inducible Nar1 gene is up-regulated, and protein content and enzyme activity also increase in leaves of barley seedlings treated with ALA. We suggest that one of the mechanisms of ALA-enhanced growth and development of barley plants is regulation of NR at the transcriptional and translational levels.

Application of 5-aminolevulinic Acid for the in vitro Micropropagation of Cymbidium as a Potential Novel Plant Regulator

Application of 5-aminolevulinic Acid for the in vitro Micropropagation of Cymbidium as a Potential Novel Plant Regulator

Role of 5-aminolevulinic acid in the salinity stress response of the seeds and seedlings of the medicinal plant Cassia obtusifolia L.

BackgroundSoil salinity, one of the major abiotic stresses affecting germination, crop growth, and productivity, is a common adverse environmental factor. The possibility of enhancing the salinity stress tolerance of Cassia obtusifolia L. seeds and seedlings by the exogenous application of 5-aminolevulinic acid (ALA) was investigated.ResultTo improve the salinity tolerance of seeds, ALA was applied in various concentrations (5, 10, 15, and 20 mg/L). To improve the salinity tolerance of seedlings, ALA was applied in various concentrations (10, 25, 50, and 100 mg/L). After 10 mg/L ALA treatment, physiological indices of seed germination (i.e., germination vigor, germination rate, germination index, and vigor index) significantly improved. At 25 mg/L ALA, there was a significant protection against salinity stress compared with non-ALA-treated seedlings. Chlorophyll content, total soluble sugars, free proline, and soluble protein contents were significantly enhanced. Increased thiobarbituric acid reactive species and membrane permeability levels were also inhibited with the ALA treatment. With the treatments of ALA, the levels of chlorophyll fluorescence parameters, i.e., the photochemical efficiency of photosystem II (Fv/Fm), photochemical efficiency (Fv'/Fm'), PSII actual photochemical efficiency (ΦPSII), and photochemical quench coefficient (qP), all significantly increased. In contrast, the non-photochemical quenching coefficient (NPQ) decreased. ALA treatment also enhanced the activities of superoxide dismutase, peroxidase, and catalase in seedling leaves. The highest salinity tolerance was obtained at 25 mg/L ALA treatment.ConclusionThe plant growth regulator ALA could be effectively used to protect C. obtusifolia seeds and seedlings from the damaging effects of salinity stress without adversely affecting plant growth.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-18) contains supplementary material, which is available to authorized users.

Clearance mechanism of protoporphyrin IX from mouse skin after application of 5-aminolevulinic acid

5-Aminolevulinic acid (ALA) or its esters mediated photodynamic therapy (PDT) is the most widely practiced form of PDT in dermatology. One of its advantages is that undesirable photosensitization lasts only for 24-48 h. In order to optimize ALA-PDT it is necessary to understand the mechanisms of intracellular production and clearance of PpIX (efflux from cells into blood stream and/or its conversion into haem). The aim of this study is to investigate the factors controlling the clearance of intracellular PpIX from healthy skin of mice. PpIX was induced in mouse skin by topical or systemic application of ALA or by topical application of the iron chelator ethylenediaminetetraacetic acid (EDTA). Fluorescence spectroscopy was used to study PpIX kinetics in alive and dead skin. Topical application of ALA or EDTA leads to porphyrin production in living skin, but not in excised skin. The clearance rates of PpIX from alive and dead skin were the same in the absence of an intracellular ALA pool. The clearance half-life of EDTA-induced PpIX was 4-7 times longer than that of PpIX after application of ALA. Skin temperature and intracellular iron availability strongly affect PpIX clearance, while ALA application mode (topical versus systemic) and skin viability (dead versus alive) have no influence on PpIX decay. These results demonstrate that the clearance kinetics of PpIX from skin are determined mostly by the conversion of PpIX into haem, while the cellular efflux of PpIX into blood plays a minor role.

5-Aminolevulinic acid enhances photosynthetic gas exchange, chlorophyll fluorescence and antioxidant system in oilseed rape under drought stress

This study evaluates the role of exogenous foliar application of 5-aminolevulinic acid (ALA) on water relations, gas exchange, chlorophyll fluorescence, and the activities and gene expression patterns of antioxidant enzymes in leaves of oilseed rape under drought stress and recovery conditions. Seedlings at four-leaf stage were imposed to well-watered condition (80 % of water-holding capacity) or drought stress (40 % of water-holding capacity) and subsequently foliar sprayed with water or ALA (30 mg l−1). Drought suppressed the accumulation of plant biomass and decreased chlorophyll content and leaf water status (relative water content and water potential). The actual quantum yield of photosystem II and electron transport rates were hampered in parallel to net photosynthetic rate. However, drought stress induced the accumulation of malondialdehyde (MDA) and hydrogen peroxide, enhanced the activities of catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase and up-regulated the expression of APX and GR. After rehydration for 4 days, the growth of drought-treated seedlings was restored to normal level for most of the physiological parameters. Foliar application of ALA maintained relatively higher leaf water status and enhanced chlorophyll content, net photosynthetic rate, actual quantum yield of photosystem II, photochemical quenching, non-photochemical quenching and electron transport rates in stressed leaves. Exogenous ALA also alleviated the accumulation of MDA and hydrogen peroxide, increased the activities of antioxidant enzymes and enhanced the expression of CAT and POD in drought-treated plants. These results indicate that ALA may effectively protect rapeseed seedlings from damage induced by drought stress.

Needle-free injection of 5-aminolevulinic acid in photodynamic therapy for the treatment of condylomata acuminata

The external application of 5-aminolevulinic acid (ALA) in photodynamic therapy (PDT) results in a shallow penetration depth in thick or extensive condylomata acuminata (CA) lesions, thus demonstrating a poor therapeutic effect for those patients. To compare the efficacy of needle-free injection with external application of ALA in PDT for the treatment of CA, 160 CA patients with thick or extensive warts received ALA-PDT by means of external application or needle-free injection of ALA, respectively. The complete response (CR) rate and recurrence rate in the two groups were analyzed. The CR rate after the first treatment in the needle-free injection group (68.8%) was significantly higher compared with that in the external application group (52.5%; P=0.035). The recurrence rates in the needle-free injection group and external application group were 4.1 and 15.4%, respectively (P=0.022). The needle-free injection of ALA increases the therapeutic effect of PDT for CA patients with thick or extensive lesions. It shortens the treatment time and reduces the recurrence rate, and has great potential in the treatment of CA.

The possibility of using 5-aminolevulinic acid in lead phytoextraction process

Studies conducted in the greenhouse involving a sunflower (Helianthus annuus L.) grown in medium containing 200 mg Pb ∙ dm-3 which were designed to evaluate the impact of foliar nutrition with 5-aminolevulinic acid (5-ALA) on the chemical composition of sunflower in reference to possible use of this plant in the process of induced phytoextraction. The study revealed from 4.8% to 34.1% increase in aboveground matter of sunflower which was sprayed by 5-ALA solution at concentrations of 0.01-0.1 ppm in comparison with plants grown in a medium containing 200 mg Pb ∙ dm-3 with no 5-ALA spraying. The sunflower leaves sprayed by 5-ALA solution contained from 3.78% to 27.1% more lead in comparison to plants not sprayed by this agent. As well as remarkable decrease in lead content from 17.4 to 33.4% was recorded in the roots of sunflower sprayed by 5-ALA solution. The lead content in plant shoots was independent from foliar application of 5-aminolevulinic acid.

Rheological characterization and permeation behavior of poloxamer 407-based systems containing 5-aminolevulinic acid for potential application in photodynamic therapy

Topical application of 5-aminolevulinic acid (ALA) in photodynamic therapy is of great interest because of avoiding systemic side effects with such an easy way of application. However, due to ALA's high polarity its dermal bioavailability is rather limited and thus, permeation enhancement of this active is of major interest in research. In a previous study, a semisolid poloxamer 407-based (POX), five-component system ("thermogel") was developed for permeation enhancement of ALA across isolated human stratum corneum. In the present study, five-component systems of systematically varied compositions were investigated both rheologically and in terms of permeation enhancement. The five-component systems contained water, a fixed combination of 1:1 of isopropyl alcohol (IPA) and dimethyl isosorbide (DMIS) and a fixed ratio of 4:1 of POX to propylene glycol dicaprylocaprate (MIG). Rheological characterization showed that complex viscosity depended on IPA/DMIS and POX/MIG content. The gelation temperature (GT) was strongly influenced by interactions between MIG, IPA and DMIS. Regarding permeation behavior, several systems showing better permeation fluxes than the original "thermogel" were identified. Surprisingly, permeation flux did not inversely correlate with the complex viscosity, showing that permeation behavior may depend on a variety of further physicochemical characteristics including individual composition and microstructure of the respective formulation.

Fluorescence-Guided Operation in Recurrent Glioblastoma Multiforme Treated with Bevacizumab—Fluorescence of the Noncontrast Enhancing Tumor Tissue?

The oral application of 5-aminolevulinic acid (ALA) leads to an accumulation of fluorescent porphyrins in malignant glioma tissue, which simplifies complete tumor resection. If pretreated with bevacizumab, a vascular endothelial growth factor (VEGF) antibody, these patients might not show a contrast enhancement on magnetic resonance imaging (MRI) despite tumor progression. As VEGF antibodies induce a normalization of the tumor vasculature, it is not known whether fluorescence-guided surgery is of any value in patients pretreated with this antibody. One might speculate that missing contrast enhancement on MRI could result in minor or missing fluorescence after the application of ALA. Attempting to give some answers, we report the case of a patient who underwent fluorescence-guided reoperation of recurrent glioblastoma multiforme pretreated with bevacizumab.

Topical application of 5-aminolevulinic acid followed by 595-nm pulsed dye laser irradiation for the treatment of recalcitrant port-wine stains: A primary study

Objective: To evaluate the effectiveness and safety of topical application of 5-aminolevulinic acid (ALA) followed by pulsed dye laser (PDL) irradiation for the treatment of recalcitrant port-wine stain (PWS). Methods: Thirty-five patients (19 females and 16 males) with recalcitrant PWS were treated with topical application of 20% ALA and then irradiated with a 595-nm PDL (energy density of 6.5–9.0 J/cm2 and pulse duration of 6 or 10 ms) at 6–8 weeks intervals. Clinical improvement of lesions was evaluated by comparing photographs of lesions at baseline and 2 months after the last treatment. Results: Topical ALA spreading followed by irradiation with a 595-nm PDL improved PWS in 21 out of 35 patients who were previously recalcitrant to PDL treatment alone. Side effects were limited to transient erythema, vesicle formation, edema and mild purpura with no obvious subsequent scarring or undesirable pigment changes. Conclusion: The topical application of ALA followed by irradiation with a 595-nm PDL is an effective and safe treatment for PWS recalcitrant to PDL therapy alone.

Fluorescence examination and photodynamic therapy of facial squamous cell carcinoma—A case report

To evaluate the usefulness and feasibility of fluorescence examination and photodynamic therapy (PDT) for facial squamous cell carcinoma. A 68-year-old male patient underwent 3 courses of PDT. The first treatment was carried out using topically applied 5-aminolevulinic acid (ALA) to the affected area (5.0 cm × 2.5 cm) 3h before light irradiation. ALA/PpIX-mediated fluorescence was used to visualize the lesion and its margin. The lesion was illuminated with a 630 nm laser at 120 J/cm². Thirty days later, the residual lesion received the second treatment at the same light dose after topical use of ALA. Despite the tumor was treated with two courses of PDT, The lesion was not cleared completely yet. At last we used topical ALA and intravenous injection of HiPorfin, with the same light dose, then the lesion was cured and the patient was tumor-free followed up for over 15 months. ALA/PpIX-mediated fluorescence visualized the lesion location. Complete cure was achieved after the third course of ALA/Hiporfin PDT. During the 15 months of followed up, no recurrence was noticed. ALA assisted fluorescence examination can be a useful tool to visualize the malignant lesion and determine its margin. ALA-PDT is effective for superficial lesions, but for thicker and deeper lesions, systemic administration of photosensitizer is indispensable.

5-Aminolevulinic acid pretreatment mitigates drought stress of cucumber leaves through altering antioxidant enzyme activity

To promote the application of 5-aminolevulinic acid (ALA) in agricultural production, a cucumber variety ( Cucumis sativus L. cv. Yuexiu no. 3) was pretreated with 3 μM ALA for 2 d and then was exposed to drought stress induced by 10% polyethylene glycol (PEG) 6000. We investigated whether exogenous ALA could protect cucumbers from drought stress and whether the protective effect was associated with the regulation of antioxidant enzymes. After 2 d of ALA pretreatment, no changes were observed in plant growth indexes including plant heights and the fresh and dry weights of shoots and roots and the second leaves. Meanwhile, we obtained the increased activities of superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7), glutathione peroxidase (GSH-Px, EC 1.11.1.9), ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2), and monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) in ALA-pretreated leaves and as well got the elevated content of reduced glutathione (GSH). When the ALA-pretreated plants were subjected to drought, the antioxidants changed further. Drought stress increased the activities of SOD, catalase (CAT, EC 1.11.1.6), GPX, GSH-Px, APX, GR, dehydroascorbate reductase (DHAR, EC 1.8.5.1) and MDHAR in leaves and also enhanced the contents of GSH and ascorbate. However, the combination of ALA pretreatment and drought enhanced the antioxidant activities even more. Moreover, drought decreased plant growth indexes, resulted in 83.3% withered leaves and increased the levels of superoxide radical (O 2 −) and hydrogen peroxide (H 2O 2) in leaves. When ALA pretreatment was combined with drought, we observed the improved growth inhibition and 48.2% withered leaves, and found the decreased levels of O 2 − and H 2O 2 in comparison to non-pretreated stressed plants. We propose that exogenous ALA can regulate antioxidant activities, which are altered further after ALA pretreatment is combined with PEG-induced drought stress; thus ALA pretreatment protects cucumbers from drought stress.

Characterization of a pseudo ternary phase diagram of poloxamer 407 systems for potential application of 5-aminolevulinic acid in photodynamic therapy

A poloxamer 407 (POX) gel containing dimethyl isosorbide (DMIS), isopropyl alcohol (IPA), propylene glycol dicaprylocaprate (MIG) and water has been suggested in a previous study for permeation enhancement of 5-aminolevulinic acid (ALA) across isolated human stratum corneum. The purpose of this study was to characterize other formulations coming from the same pseudo ternary phase diagram as the “Thermogel” in order to find out which of them show appropriate characteristics to be used as a vehicle for ALA since it could be shown that variation of the ingredients’ content had an influence on the permeation rate. A pseudo ternary phase diagram was developed with water, a fixed combination of 1:1 of IPA and DMIS and a fixed ratio of 4:1 POX to MIG. The systems were categorized according to their consistencies and ringing gel characteristics with special emphasis on appropriate formulations for dermal application. Polarizing microscopy enabled a clear differentiation between isotropic and anisotropic systems. Wide angle X-ray diffraction analyzes confirmed that anisotropy was due to crystalline POX. Furthermore both methods showed that IPA/DMIS was an inferior solvent mixture for POX related to water.

5-Aminolevulinic acid-mediated photodynamic therapy to superficial malignant skin tumors using Super Lizer

Photodynamic therapy (PDT) using topically applied 5-aminolevulinic acid (ALA) has become a generally accepted treatment modality for superficial malignant skin tumors. However, the costly excimer-dye laser, diode laser and light-emitting diode (LED) frequently used to administrate PDT are impractical to use in most dermatology clinics. This study evaluated the effectiveness of ALA-mediated PDT using a Super Lizer (Tokyo Iken, Tokyo, Japan) equipped with band-pass filters in 38 patients with superficial malignant skin tumors (33 cases of actinic keratosis and five cases of Bowen's disease). Twenty-one cases (18 cases of actinic keratosis and three cases of Bowen's disease) were successfully treated, and the other 17 cases (15 cases of actinic keratosis and two cases of Bowen's disease) showed partial remission after single or repeated administration of PDT. PDT repeated three times at weekly intervals was more effective against actinic keratosis than randomly repeated procedures. The Super Lizer is easy to handle and move, and is less expensive than other known machinery and is useful for PDT in dermatology, especially under the protocol of three times at weekly intervals for the treatment of actinic keratosis.