Biomedical subjects

T M Dubbelman

Publications and source records attributed to T M Dubbelman.

At least 19 recordsLinked to original sources

PubMed2000

5-aminolevulinic acid induced lipid peroxidation after light exposure on human colon carcinoma cells and effects of alpha-tocopherol treatment.

This work relates to studies on modes of phototoxicity by protoporphyrin (PpIX) after incubation of 5-aminolevulinic acid (5-ALA) on cultured cells. Lipid peroxidation in the 5-ALA incubated primary adenocarcinoma cells from the rectosigmoid colon (WiDr cells) was determined by measurement of protein-associated thiobarbituric acid reactive substances (TBARS). TBARS were increased 2-fold in cells treated with 2 mM 5-ALA for 3.5 h in serum enriched medium. After illumination of 5-ALA incubated cells, TBARS were formed in a light dose dependent manner. TBARS analysis were compared with high-performance liquid chromatography (HPLC) analysis of malondialdehyde, and results indicate that 90% of the thiobarbituric reactive substances were due to malondialdehyde. Pretreating WiDr cells with alpha-tocopherol for 48 h inhibits the cytotoxic effect of 5-ALA and increases 5-fold the light dose needed to kill 50% of the cells. Pretreatment with alpha-tocopherol shows a considerable decrease (about 80%) on TBARS formation after illumination. The cellular content of alpha-tocopherol was determined by HPLC and found to be 15.3 pmol/10(6) cells.

Adenocarcinoma↗

PubMed2000

Selective protection of RBCs against photodynamic damage by the band 3 ligand dipyridamole.

BACKGROUND: All studied photosensitizers for virus inactivation impair RBCs. To reduce damage to the RBCs without affecting virucidal activity, selective protection of the RBCs is necessary. The ability of the band 3 ligand, dipyridamole, to react with singlet oxygen and to increase the selectivity of photosterilization was investigated. STUDY DESIGN AND METHODS: Solutions of dipyridamole were illuminated in the presence of tetrasulfonated aluminum phthalocyanine (AlPcS(4)) and dimethylmethylene blue (DMMB). Solutions of amino acids, RBCs, and vesicular stomatitis virus (VSV) in RBC suspensions were photodynamically treated in the presence or absence of dipyridamole. RESULTS: Illumination of a solution of dipyridamole in the presence of AlPcS(4) or DMMB resulted in changes in the optical spectrum of dipyridamole. The photooxidation of dipyridamole was inhibited by azide and augmented by D(2)O, which suggests the involvement of singlet oxygen. Photooxidation of amino acids and photodamage to RBCs was strongly reduced in the presence of dipyridamole. In contrast, photoinactivation of VSV in RBC suspensions was only slightly affected by dipyridamole. CONCLUSION: Dipyridamole can improve the specificity of photodynamic sterilization of RBC concentrates, thereby increasing the practical applicability of this photodecontamination method.

Antiviral Agents↗

PubMed2000

Plasma membrane properties involved in the photodynamic efficacy of merocyanine 540 and tetrasulfonated aluminum phthalocyanine.

Merocyanine 540 (MC540)-mediated photodynamic damage to erythrocytes was strongly reduced when illumination was performed at pH 8.5 as compared to pH 7.4. This could be explained by high pH-mediated hyperpolarization of the erythrocyte membrane, resulting in decreased MC540 binding at pH 8.5. In accordance, the MC540-mediated photooxidation of open ghosts was not inhibited at pH 8.5. Photoinactivation of vesicular stomatitis virus (VSV) was not inhibited at pH 8.5. This suggests that illumination at increased pH could be an approach to protect red blood cells selectively against MC540-mediated virucidal phototreatment. With tetrasulfonated aluminum phthalocyanine (AIPcS4) as photosensitizer, damage to erythrocytes, open ghosts and VSV was decreased when illuminated at pH 8.5. A decreased singlet oxygen yield at high pH could be excluded. The AIPcS4-mediated photooxidation of fixed erythrocytes was strongly dependent on the cation concentration in the buffer, indicating that the surface potential may affect the efficacy of this photosensitizer. This study showed that altering the environment of the target could increase both the efficacy and the specificity of a photodynamic treatment.

Erythrocyte Membrane↗

PubMed1999

Photodynamically induced effects in colon carcinoma cells (WiDr) by endogenous photosensitizers generated by incubation with 5-aminolaevulinic acid.

Human adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid (5-ALA) in the presence of 10% foetal calf serum. The treatment induces a linear accumulation of protoporphyrin IX (PpIX) for at least 7.5 h. After 7.5 h of incubation about 45% of the PpIX accumulated is cell-bound, while the rest is found in the medium (25%) or lost from the cells during washing with phosphate-buffered saline (30%). Exposure to white light at an intensity of 30 W/m2 for 18 min results in 95% reduction of clonogenicity in cells treated with 2 mM 5-ALA for 3.5 h. The enzymatic activities of enzymes located in cytosol (glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase) and lysosomes (acid phosphatase and beta-glucuronidase) are not influenced by a 5-ALA and light treatment inactivating about 35% of the cells. The MTT assay, which reflects mitochondrial dehydrogenase activity, but not succinate dehydrogenase, is partly inhibited by the same treatment. Treatment with 5-ALA in the absence of light increases O2 consumption by a factor of two, while the O2 consumption is inhibited when 5-ALA treatment is combined with exposure to light. In addition, 5-ALA and light exposure enhance accumulation of rhodamine 123 by 40% and reduce the intracellular ATP level by 25%. Confocal laser scanning microscopical analysis indicates granular perinuclear localization of the PpIX formed by 5-ALA treatment. In conclusion, photodynamic treatment using 5-ALA as a prodrug induces damage to mitochondrial function without inhibiting lysosomal and cytosolic marker enzymes.

Adenosine Triphosphate↗

PubMed1999

Photodynamic treatment of adenoviral vectors with visible light: an easy and convenient method for viral inactivation.

Recombinant adenovirus vectors are popular tools for gene transfer and gene therapy. However biosafety constraints require that all handling of the vectors and vector-containing samples is restricted to dedicated containment laboratories, unless they had undergone a validated virus-inactivation procedure, which decontaminates the samples from any active virus. In this study we evaluated the feasibility of photodynamic treatment (PDT) with visible light to inactivate recombinant adenovirus vectors in biological samples, with minimum associated effects on other biological activities. Several photosensitizers were tested for their capacity to inactivate a model human adenovirus vector, AdCMVLuc, upon illumination. Four photosensitizers (methylene blue (MB), rose bengal (RB), uroporphyrin (UP) and aluminum phthalocynine tetrasulphonate (AIPcS4)) could inactivate the adenovirus, as measured by expression of the luciferase reporter gene and by plaque assay. Of these, MB demonstrated to be the most effective sensitizer in phosphate-buffered saline (PBS), giving > 7 log10 inactivation of the adenovirus. DNA isolated from MB- and light-treated virions was inefficient as a template for transcription. Furthermore, Southern blot analysis revealed fragmentation of the viral DNA. Based on its preference for DNA, MB is suited for adenovirus inactivation in blood plasma. Spiking experiments in which AdCMVLuc was added to plasma samples demonstrated a reduction (> 4 log10-fold) of reporter gene expression to almost background levels. In contrast to MB, photodynamic treatment with RB, UP or AIPcS4 did not lead to DNA damage. Although alterations of the viral capsid could not be detected, the binding pattern of the particles to target cells was significantly changed. Taken together, our data demonstrate that PDT is an efficient, convenient and useful method for the inactivation of adenovirus vectors in biological samples.

Adenoviridae↗

PubMed1999

Photodynamic sterilization of red cells and its effect on contaminating white cells: viability and mechanism of cell death.

BACKGROUND: Phthalocyanines are useful sensitizers for photodynamic sterilization of red cell concentrates. Various lipid-enveloped viruses can be inactivated with only limited red cell damage. Because white cells are involved in the immunomodulatory effects of blood transfusions, the study of the effect of photodynamic treatment on these cells is imperative. STUDY DESIGN AND METHODS: White cell-enriched red cell suspensions were photodynamically treated with either the hydrophobic Pc4 (HOSiPcOSi-(CH3)2(CH2)3N(CH3)2) or water-soluble aluminum phthalocyanine tetrasulfonate (AIPCS4) under virucidal conditions. Viability of white cell subpopulations on Days 0, 1, and 4 after treatment was determined by fluorescence-activated cell sorting by flow cytometric analysis of propidium iodide uptake. Apoptosis induction was studied by DNA ladder formation and staining for an early marker of apoptosis (annexin V). RESULTS: Treatment with Pc4 causes a significant decrease in cell viability of all white cells, as shown by prodidium iodide uptake. Monocytes and granulocytes are the most sensitive, and lymphocytes are relatively more resistant. Some of the cells die by apoptosis, which is induced within 30 minutes after treatment. Treatment with AIPCS4 damages only monocytes; other cell populations are not affected. CONCLUSIONS: Physicochemical properties of the photosensitizers partly determine their effect on white cells. Differences in intracellular localization are likely to be responsible for the effects observed.

Annexin A5↗

PubMed1999

Transfusion-transmitted diseases: risks, prevention and perspectives.

During the past decades major improvements in blood safety have been achieved, both in developed and developing countries. The introduction of donor counseling and screening for different pathogens has made blood a very safe product, especially in developed countries. However, even in these countries, there is still a residual risk for the transmission of several pathogens. For viruses such as the human immunodeficiency virus (HIV), and the hepatitis viruses B and C, this is due mainly to window-period donations. Furthermore, the threat of newly emerging pathogens which can affect blood safety is always present. For example, the implications of the agent causing new variant Creutzfeld-Jakob disease for transfusion practice are not yet clear. Finally, there are several pathogens, e.g. CMV and parvo B19, which are common in the general donor population, and might pose a serious threat in selected groups of immunosuppressed patients. In the future, further improvements in blood safety are expected from the introduction of polymerase chain reaction for testing and from the implementation of photochemical decontamination for cellular blood products. The situation in transfusion medicine in the developing world is much less favorable, due mainly to a higher incidence and prevalence of infectious diseases.

Bacterial Infections↗

PubMed1999

Inhibition of various steps in the replication cycle of vesicular stomatitis virus contributes to its photoinactivation by AlPcS4 or Pc4 and red light.

Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AlPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene-labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AlPcS4 treatment, which correlated with the decrease in RNA synthesis. For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AlPcS4 treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8-oxo-7,8-dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.

Animals↗

PubMed1999

Oxidized low-density lipoprotein as a delivery system for photosensitizers: implications for photodynamic therapy of atherosclerosis.

Photodynamic therapy is a promising new strategy in the treatment of cardiovascular diseases. Photodynamic therapy for vascular diseases may be improved by the specific delivery of photosensitizers to the atherosclerotic lesion. In this study, we studied whether oxidatively modified low-density lipoprotein (OxLDL) could be used as a specific carrier for photosensitizers, thereby using the scavenger receptor expressed on macrophages as a target. The photosensitizer aluminum phthalocyanine chloride (AlPc) was incorporated into OxLDL, and its photodynamic effects were studied. Macrophages (RAW 264.7) were incubated with various concentrations of OxLDL-AlPc for different periods. After illumination of the cells with red light, cytotoxicity was observed that was dependent on the time of illumination and incubation. Macrophages incubated with OxLDL-AlPc that were not illuminated revealed no cytotoxicity. The uptake of the OxLDL-AlPc complexes was mediated by scavenger receptors expressed on macrophages. In the presence of the polyanion polyinosinic acid, a specific ligand for scavenger receptors, no cytotoxicity could be observed. Serum incubations of the OxLDL-AlPc complexes revealed that these complexes stay intact after incubation. No redistribution of AlPc to other plasma (lipo-) proteins could be detected, and 80-90% of the AlPc remained associated with the OxLDL particle. These results indicate that OxLDL may function as a specific delivery system for photosensitizers to the scavenger receptors expressed on the macrophages in the atherosclerotic lesion, increasing the beneficial effects of photodynamic therapy for cardiovascular diseases.

Animals↗

PubMed1998

The characterisation of three substituted zinc phthalocyanines of differing charge for use in photodynamic therapy. A comparative study of their aggregation and photosensitising ability in relation to mTHPC and polyhaematoporphyrin.

Three substituted zinc (II) phthalocyanines (one anionic, one cationic and one hydrophobic) have been compared to two clinically used photosensitisers, 5,10,15,20-tetra (m-hydroxyphenyl) chlorin (mTHPC) and polyhaematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Oxygen-consumption experiments, performed to follow the photo-oxidation of tryptophan, histidine and bovine serum albumin (BSA), suggest that the anionic phthalocyanine is the most efficient photosensitiser. The efficacy of BSA oxidation is much greater than that of tryptophan or histidine, which is partly due to monomerisation of the sensitisers upon binding to BSA. Spectra recorded in aqueous solution reveal that all five compounds are highly aggregated, but monomerisation is induced upon the addition of BSA or methanol. Using a range of methanol-buffer solutions, the aggregation state has been directly related to the efficacy of tryptophan photo-oxidation with maximal rates of oxidation achieved when the sensitiser is monomeric. Using erythrocytes as a simple membrane model, the efficacy of each sensitiser exhibits a different trend from that predicted by oxygen-consumption experiments. The anionic phthalocyanine is the least effective at photohaemolysis, whereas the cationic and hydrophobic phthalocyanines have improved activity over PHP and mTHPC.

Animals↗

PubMed1998

Effect of membrane potential on the binding of merocyanine 540 to human erythrocytes.

Illumination of erythrocytes in the presence of merocyanine 540 (MC540) resulted in changed binding characteristics of MC540, i.e. a red shift in the emission maximum of bound dye with an increase in the relative fluorescence quantum yield. Aluminum phthalocyanine tetrasulfonate-mediated photodynamic treatment, before addition of MC540, resulted in a comparable change in the MC540-binding characteristics with, in addition, an increase in the concentration of MC540 in the membrane. Both photodynamic treatments induce depolarization of the red cell membrane, with a dose dependency comparable to that of changed MC540 binding. Also depolarization, induced by incubation of the cells with A23187 in the presence of Ca2+ in high [K+] buffer, resulted in similar changes in the MC540 binding characteristics. These results indicate a relation between photodynamically induced membrane depolarization and changed MC540-binding characteristics. Hyperpolarization induced by incubation with A23187 in low [K+] buffer resulted in decreased binding of MC540. In accordance, the MC540-mediated photodamage to red cells decreased upon hyperpolarization of the cells. The results indicate that the binding of MC540 to erythrocytes is strongly dependent on the membrane potential and that hyperpolarization of the membrane could be a possible protection mechanism for erythrocytes against MC540-mediated photodynamic damage.

Dose-Response Relationship, Radiation↗

PubMed1997

Decreased expression of HLA class I on ocular melanoma cells following in vitro photodynamic therapy.

Our objective was to investigate whether photodynamic therapy (PDT) influences the expression of HLA Class I and beta 2-microglobulin molecules on cultured uveal melanoma cells. Uveal melanoma cells were incubated with hematoporphyrin esters (HPE) and illuminated using red light. HLA expression on cells was determined by flowcytometry. PDT treatment induced an immediate reduction in expression of HLA Class I and beta 2-microglobulin, followed by a transient increase in expression after 2 h. Normalization occurred after 6 h. Treatment of ocular melanoma cells with PDT temporally alters the expression of HLA Class I and beta 2-microglobulin, which may affect anti-tumor-immune responses.

Antigens, Neoplasm↗

PubMed1997

Effect of hydrogen peroxide on the binding of Merocyanine 540 to human erythrocytes.

The fluorescent dye Merocyanine 540 (MC540) is often used as a probe to monitor the molecular packing of phospholipids in the outer leaflet of biomembranes. In a previous study we showed that the increased staining of erythrocytes with a perturbed membrane structure was mainly due to an increase in the fluorescence yield of cell-bound MC540, rather than to an increase of the number of bound molecules. Erythrocytes and ghosts exposed to continuous fluxes of H2O2 exhibited pronounced lipid peroxidation. Further, red blood cells subjected to this form of oxidative stress also showed increased staining with MC540. It appeared that this was caused by a strong increase in binding of MC540, together with a slight red shift of the fluorescence emission maximum and a small increase in the fluorescence yield of bound MC540. The changed MC540 binding characteristics were not observed when lipid peroxidation was suppressed by the presence of the antioxidant BHT in the incubation medium. However, open ghosts exposed to H2O2 showed no increase of MC540 binding, excluding a direct involvement of lipid peroxidation. Measurement of fluorescence emission spectra and gel filtration studies showed that MC540 can bind to H2O2-exposed hemoglobin. Experiments with erythrocytes lysed in hypotonic medium after exposure to H2O2 revealed that peroxidation of lipids with H2O2 induced a non-specific permeabilization of the plasma membrane to MC540, thereby allowing MC540 to bind to the oxidatively denatured, more hydrophobic hemoglobin. These results indicate that conclusions about packing of phospholipids in the outer leaflet of the membrane based on increased MC540-staining should be drawn with care.

Adolescent↗

PubMed1997

Uric acid and ascorbic acid redox ratios in plasma and tracheal aspirate of preterm babies with acute and chronic lung disease.

This study compared plasma redox ratios of uric acid and ascorbic acid in well preterm babies with those with respiratory distress syndrome (RDS) and chronic lung disease (CLD), and investigated the relationship between these ratios and their respective measurements in tracheal aspirate. On day 1 after birth, plasma allantoin and allantoin/uric acid ratio were elevated in CLD (p < .05), and both markers of oxidative stress enabled early prediction of development of CLD (sensitivity and specificity: 54 and 83%, respectively). The relation between allantoin production and oxidative stress is supported by the correlation between the allantoin level and oxygen therapy in both RDS and CLD (p < .05). Reduced and oxidize ascorbic acid in plasma decreased postnatally in all groups and their redox ratio remained stable. Uric acid and ascorbic acid redox ratios were significantly elevated in tracheal aspirates compared to plasma samples (p < .05), and there was a strong positive correlation between both ratios (p < .005). These markers may be useful in monitoring babies with respiratory distress.

Antioxidants↗

PubMed1997

Single strand breaks and mutagenesis in yeast induced by photodynamic treatment with chloroaluminum phthalocyanine.

Photodynamic treatment of the yeast Kluyveromyces marxianus with the sensitizer aluminum phthalocyanine results in loss of clonogenicity. In this paper the effect of this treatment on DNA of this yeast was investigated by searching for single strand breaks and forward mutations. Using the alkaline step elution technique it was found that illumination of the yeast in the presence of aluminum phthalocyanine resulted in an increase in single strand breaks. These could, partially, be repaired by post-incubating illuminated cells in growth medium. At comparable survival levels, photodynamic treatment with aluminum phthalocyanine induced fewer single strand breaks than X-ray treatment. By using a medium containing 5-fluoroorotic acid, mutants in the uracil biosynthetic pathway were selected. Photodynamic treatment resulted in a light dose dependent increase of the mutation frequency. The observed mutagenicity of photodynamic treatment of the yeast with phthalocyanine was lower than the mutagenicity of UVC and X-ray treatment at equal colony forming capacity, indicating that photodynamic treatment is the least mutagenic of those treatments. It is concluded that photodynamic treatment of K. marxianus results in DNA damage. Saccharomyces cerevisiae rad14 and rad52 mutants were used to determine the effect of the nucleotide excision repair and recombinational repair pathways, respectively, on survival after photodynamic treatment. Our data indicate that DNA damage is not the main determinant for cell killing by photodynamic treatment and that the type of damage induced is apparently not subject to RAD14- or RAD52 controlled repair.

Aluminum↗

PubMed1997

Shelf-life of photodynamically sterilized red cell concentrates with various numbers of white cells.

BACKGROUND: Phthalocyanines are useful sensitizers for the photodynamic sterilization of red cell concentrates. The use of the phthalocyanine Pc4 (HOSiPcOSi(CH3)2(CH2)3N(CH3)2) and red light is very efficient in killing various viruses. The addition of scavengers of Type I photodynamic reactions and the use of cremophor to deliver Pc4 give protection to the red cells. STUDY DESIGN AND METHODS: Various red cell components, either white cell-enriched, buffy coat-removed, or white cell-reduced, have been used to study the effect of photodynamic treatment with Pc4 on hemoglobin and potassium leakage and on ATP and glucose levels after prolonged storage. RESULTS: After treatment, storage interval-dependent damage to the red cells could be observed. In components with 26 x 10(9) white cells per L, virus inactivation was less efficient than that in components with no or 2 x 10(9) white cells per L. Similarly, red cells were less affected by the treatment in components with a large number of white cells. Pretreatment storage and use within 1 week after photodynamic treatment induce less damage to the red cells at the moment of transfusion. CONCLUSION: Various improvements in the treatment protocol may ultimately lead to the implementation of photodynamic treatment in transfusion practice. In this respect, the white cell content of the red cell concentrates should be taken into account.

Adenosine Triphosphate↗

PubMed1997

Electron spin resonance evidence of the generation of superoxide anion, hydroxyl radical and singlet oxygen during the photohemolysis of human erythrocytes with bacteriochlorin a.

Photodynamic therapy with bacteriochlorin a (BCA) as sensitizer induces damage to red blood cells in vivo. To assess the extent of the contributuion of reactive oxygen species (ROS) and to determine a possible reaction mechanism, competition experiments with assorted ROS quenching or/and enhancing agents were performed in human erythrocytes as model system and in phosphate buffer. In the erythrocyte experiments, a 2% suspension was incubated with BCA for 1 h, washed with phosphate-buffered saline, resuspended and subsequently illuminated with a diode laser using a fluence rate of 2.65 mW/cm2. Potassium leakage and hemolysis were light and BCA dose dependent. Adding tryptophan (3.3 mM), azide (1 mM) or histidine (10 mM) to the erythrocyte suspension before illumination delayed the onset of K-leakage and hemolysis suggesting a type II mechanism. The D2O did not affect K-leakage nor photohemolysis. Adding mannitol (13.3 mM) or glycerol (300 nM) also caused a delay in the onset of K-leakage and hemolysis, suggesting the involvement of radicals. In phosphate buffer experiments, it was shown using electron spin resonance (ESR) associated with spin-trapping techniques that BCA is able to generate O2-. and OH. radicals without production of aqueous electron. Visible or UV irradiation of the dye in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct DMPO-OH. Addition of ethanol or sodium formate produced supplementary hyperfine splittings due to the respective CH3CHOH. and CO2-. radical adducts, indicating the presence of free OH.. Production of DMPO-OH was partly inhibited by superoxide dismutase (SOD), catalase and desferrioxamine, suggesting that the iron-catalyzed decomposition of H2O2 was partly involved in the formation of one part of the observed OH.. The complementary inhibition of DMPO-OH production by azide and 9,10-anthracenedipropionic acid (ADPA) was consistent with 1O2 production by BCA followed by reaction of 1O2 with DMPO and decay of the intermediate complex to form DMPO-OH and free OH.. All our results seem to indicate that BCA is a 50%/50% type 1/type 2 sensitizer in buffered aqueous solutions and confirmed that the dye-induced hemolysis of erythrocytes was cell caused by a mixed type 1/type 2 mechanism.

Cyclic N-Oxides↗

PubMed1997

In vitro fluence rate effects in photodynamic reactions with AIPcS4 as sensitizer.

It has been shown previously that the efficiency of photodynamic therapy (PDT) both in vivo and in vitro is dependent on fluence rate. In this study, different in vitro experiments showed that tetrasulfonated aluminum phthalocyanine (AIPcS4) is more efficient in photosensitization if the light is delivered at low fluence rate. Erythrocyte damage, virus inactivation and photooxidation of reduced glutathione (GSH) and histidine were all enhanced if light was delivered at 100 W/m2 as compared to 500 W/m2. Bleaching did not occur under these conditions. Oxygen depletion, shown to be important in fluence rate effects observed in vivo, does not seem to be involved. On theoretical grounds saturation of the triplet state is not likely under these conditions. A possible explantation for the observed fluence rate effects might be found in different reaction pathways, that are favored under high or low fluence rate illuminations. These reactions might involve uni- or bimolecular reactions of intermediate products, resulting in less efficiency at higher fluence rate. It proves to be important, under all circumstances, to monitor fluence rate, because a change in fluence rate, even with similar total fluences, might influence photobiological results in an unexpected way.

Erythrocytes↗