Fluorescence Excitation Spectrum of Bilirubin in Blood: A Model for the Action Spectrum for Phototherapy of Neonatal Jaundice

A recent report (Lamola et al. 2013 Pediatric Research, 74, 54–60) presents a semiempirical model for facile calculation of an action spectrum for bilirubin photochemistry in vivo using the most current knowledge of the optics of neonatal skin. The calculations indicate that competition for phototherapy light by hemoglobin in the skin is the predominant factor that defines the spectrum of light absorbed by bilirubin. If the latter is correct, a valid physical analog of the calculated spectrum is the excitation spectrum of bilirubin in blood. The fluorescence excitation spectrum was recorded and, indeed, found to be very similar to the calculated spectrum. Both spectra exhibit maxima near 476 nm and widths at half height of about 50 nm. This result supports the conclusion that light between 460 and 490 nm is most effective for phototherapy of neonatal jaundice.     

BILIRUBIN PHOTOTOXICITY TO HUMAN CELLS BY GREEN LIGHT PHOTOTHERAPY in vitro

Phototherapy of newborn infants with blue or green light is the most common treatment of neonatal hyperbilirubinemia. Using bilirubin bound to human lymphoid and basal skin cells we obtained the green light dose dependency of the bilirubin phototoxicity to these cell types. Cells (3–5× 10⁶/mL) were incubated with bilirubin complexed to human serum albumin (final concentrations 340 μM bilirubin, 150 μM albumin). Under these conditions all cells showed maximum binding of bilirubin. Irradiation with broadband green light (Λ_max= 512 nm) over 24 h led to a light dose-dependent population of cells, which contained no bilirubin on the cell membrane as determined by Nomarski interference microscopy. The light-induced mechanism of the disappearance of bilirubin caused lethal membrane damage to the cells (trypan blue exclusion test). The cell kill rate increased with the irradiation dose and with the fraction of cells with no bilirubin. When 90% of lymphoid cells were bilirubin free, 46% of them were dead (using 480 J cm^?1 green light). Similar results were obtained with basal skin cells. In addition, bilirubin-induced damage of cell membrane and nuclear membrane was also shown by transmission electron microscopy. Bilirubin (340 μM) in the dark led to 5% of the cells being killed. Basal skin cells bind 2.5 times more bilirubin molecules than lymphoid cells and showed a different bilirubin disappearance. Irradiation of bilirubin in carbon tetrachloride with 514.5 nm laser light showed generation of singlet oxygen via its luminescence at 1270 nm. These results demonstrate that green light phototherapy of hyperbilirubinemia may cause both skin and immune system damage.     

QUANTUM YIELDS FOR LASER PHOTOCYCLIZATION OF BILIRUBIN IN THE PRESENCE OF HUMAN SERUM ALBUMIN. DEPENDENCE OF QUANTUM YIELD ON EXCITATION WAVELENGTH

The quantum yield for laser photocyclization of bilirubin to lumirubin in the presence of human serum albumin (phi LR) was measured at five monochromatic excitation wavelengths in the range 450-530 nm. Solutions used were optically thin throughout the wavelength range and precautions were taken to exclude contributions from photocyclization of bilirubin XIII alpha impurities. The values obtained (7.2-18 x 10(-4] were lower than those previously reported and showed the following wavelength dependence: 457.9 less than 488.0 less than 501.7 less than 514.5 approximately equal to 528.7. However, the rate of lumirubin formation, normalized to constant fluence, decreased with wavelength over the same wavelength range and no evidence was found that photoisomerization of bilirubin to lumirubin is faster with green (514.5 or 528.7 nm) than with blue (457.9 or 488.0 nm) light. The stereoselectivity of the configurational isomerization of bilirubin to 4Z,15E and 4E,15Z isomers also was studied. This reaction became less regioselective for the 4Z,15E isomer with increasing wavelength. The observed wavelength dependence of phi LR and of the [4E,15Z]: [4Z,15E] ratio at photoequilibrium are consistent with an exciton coupling model in which intramolecular energy transfer can occur between the two pyrromethenone chromophores of the bilirubin molecule in the excited state. Relative rates of lumirubin formation in vivo at different excitation wavelengths and constant fluence were estimated for different optical thicknesses and for different skin thicknesses. These estimates suggest that the recently reported clinical equivalence of blue and green phototherapy lights probably reflects the marked variation of skin transmittance with wavelength more than wavelength-dependent photochemistry. The calculations also indicated that the optimal wavelength for phototherapy is probably on the long wavelength side of the bilirubin absorption maximum.     

CONFIGURATIONAL PHOTOISOMERIZATION OF BILIRUBIN IN VITRO–II. A COMPARATIVE STUDY OF PHOTOTHERAPY FLUORESCENT LAMPS AND LASERS

Abstract— A comparative investigation of configurational photoisomerization of bilirubin (Z.Z-BR) bound to human serum albumin (HSA) in vitro produced by fluorescent lamps currently employed in phototherapy of neonatal jaundice has been carried out by using a fast absorbance technique. Photoequilibrium Z, E-BR concentrations and rise-times are reported. Similar measurements effected with monochromatic laser lines in the blue-green spectral region are presented for comparison purposes.
Narrow-spectrum fluorescent lamps (violet, special-blue, filtered-green) produce Z,E-BR concentrations at photoequilibrium almost equal to those obtained with monochromatic lines. The photoequilibrium rise-times, however, strongly depend on the spectral bandwidth of the excitation light. Special-blue, daylight and green lamps produce Z,E-BR concentrations = 34,31, and 12%, respectively. For green lamps, the intense UV-blue-Hg lines are responsible for more than 50% of Z,E-BR concentration. Green light is found to be quite ineffective in forming Z,E isomers and very efficient in causing Z,E-BR to revert to native Z.Z-BR. Moreover, simultaneous irradiation of BR solution with special-blue and green fluorescent lamps produces almost complete inhibition of the Z→E reaction at suitable green/blue intensity ratio.
On the basis of these results a possible mechanism is presented to explain the clinical success of fluorescent green lamp phototherapy and the greater production of structural BR isomers recently reported for green than that for white lamps.     

Bilirubin photoisomerization products in serum and urine from a Crigler-Najjar type I patient treated by phototherapy

The relative compositions of the photoisomers of bilirubin-1X alpha (4Z, 15Z-bilirubin) in serum and urine of a patient with Crigler-Najjar type I syndrome treated by phototherapy are reported. High-performance liquid chromatography analysis reveals the presence of high serum levels of the configurational bilirubin photoisomer (4Z,15E-bilirubin) before the beginning of phototherapy (between 12 and 16% of the total bilirubin). The configurational photoisomer value increases during phototherapy with blue fluorescent lamps up to a photoequilibrium of about 25%, similar to that obtained in a bilirubin solution in vitro irradiated by the same lamps. This evidence suggests an inefficient serum excretion of the 4Z,15E-bilirubin. Indeed, its average half-life in serum of the Crigler-Najjar patient is found to be about 8 h. No detectable traces of the bilirubin structural isomer, lumirubin, are found in the serum. On the other hand, lumirubin represents the dominant bilirubin isomer excreted in the urine, as both 15Z and 15E configurations. Smaller amounts of 4Z,15E-bilirubin, 4E,15Z-bilirubin and native 4Z,15Z-bilirubin are observed in urine. The presence in urine of 4Z,15Z-bilirubin is probably due to a fast reversion of the configurational photoisomers to their native form. The half-life of the configurational photoisomers in urine kept at 38 degrees C is found to be of the order of a few minutes. Our study indicates that in Crigler-Najjar type I patients, mechanisms exist to excrete all bilirubin photoisomers. The lumirubin pathway seems to contribute markedly to bilirubin excretion in the urine, as occurs in jaundiced babies under phototherapy. However, the contribution of configurational isomers cannot be neglected.     

Two-photon absorption of photochromic diarylethene and its application to rewritable holographic recording

A new photochromic diarylethene (1a) has been prepared. Both its photochromic behavior and nonlinear optical properties are investigated. 1a shows excellent ring-opening (λ_max = 386 nm) and ring-closing (λ_max = 652 nm) photoisomerization with UV-Vis light irradiation. With 800 nm femtosecond pulsed laser irradiation, 1a shows two-photon-induced photoisomerization, and a two-photon absorption cross-section (σ = 423×10⁵⁰ cm⁴·s per photon) is obtained by using two-photon induced fluorescence method. The applications of two-photon absorption of 1a to holographic recording has been also investigated. A two-photon induced micro-pattern is recorded on the diarylethene 1a-PMMA film with an femtosecond laser of 800 nm, 100 fs, 1 kHz, 50 mW.     

Optimization of thermostated electrodeless discharge lamps for analytical atomic spectrometry

Thermostated and unthermostated electrodeless discharge lamps (EDL's) operated at 2450 MHz with either an A-antenna or a $\text{3}\text{4}$-wavelength Broida cavity have been critically studied with respect to the effect of type and pressure of fill gas, lamp temperature, microwave power and the form of element and/or compound within the lamp, upon the source radiant output, atomic absorption and atomic fluorescence signals of Zn-213·8 nm, Pb-283·3 nm, Mn-279·5 nm, Hg-253·7 nm and Tl-377·6 nm. Temperature control of electrodeless discharge lamps eliminates most of the problems which have previously plagued their operation. Also as a result of these studies, certain misconcepts in the operation and characteristics of electrodeless discharge lamps have been clarified; e.g., the ‘skin effect’ is present in many electrodeless discharge lamps, but all lamps also exhibit a homogeneous glow discharge; at high microwave powers and/or at high lamp temperatures, spectral lines apparently exhibit little self-absorption and self-reversal; and most important, control of lamp temperature is the most critical parameter controlling spectral output because atomization within the lamps is predominantly thermal in origin. Also, contrary to the conclusions reached by some workers evaluating the analytical usefulness of atomic fluorescence flame spectrometry, it is shown that detection limits one-to-three orders of magnitude lower result when thermostated EDL's are used in atomic fluorescence spectrometry with C₂H₂-air flames.     

PHOTOPRODUCTS OF CHLORPROMAZINE WHICH CAUSE RED BLOOD CELL LYSIS

A mechanism for chlorpromazine (CPZ) phototoxicity has been proposed that attributes the response to formation of stable, toxic photoproducts which cause cell membrane disruption. We have characterized these toxic photoproducts as dimers and higher multimers of CPZ. Chlorpromazine solutions (3 or 10 mA/) were irradiated with a medium pressure Hg lamp filtered to exclude λ < 280 nm. Five low mol wt photoproducts were separated by high performance liquid chromatography. Two were identified as CPZ-sulfoxide and promazine. Higher mol wt photoproducts were separated by Sephadex G-50 chromatography into 3 broad bands which were characterized by their absorption and fluorescence spectra. Band A (mol wt > 800) had λ_max^abs= 263 nm, λ_max^fl= 490 nm and Band B (mol wt = 350-800) had λ_max^abs= 255 nm, λ_max^fl= 450 nm. Based on the mol wt of CPZ, Band A contained trimers and higher mol wt compounds and Band B was composed of dimeric structures. BandC(λ_max^abs= 255,310 nm; λ_max^fl= 445 nm) was composed of CPZ (mol wt = 315) and the low mol wt photoproducts. Red blood cell lysis was used as an assay for the ability of photoproducts to cause membrane disruption. Bands A and B, but not Band C, caused cell lysis. These data indicate that the CPZ photoproducts which cause cell membrane disruption are dimers (Band B) and higher multimers (Band A).     

Photostimulated changes in WO3 nanosized films

During the irradiation of WO₃ films d = 7–160 nm thick by light at λ = 320 nm (I = (1.5–7) × 10¹⁵ quantum cm⁻² s⁻¹), absorption band at λ = 850 nm appeared along with absorption band edge shift to shorter waves. The subsequent irradiation of samples at λ = 850 nm caused the disappearance of the longwave absorption band. The intrinsic absorption edge of WO₃ films was determined (λ = 320 nm). The degree of transformations of WO₃ films increased under atmospheric conditions as the intensity of incident light and the time of irradiation (1–140 min) grew and as film thickness decreased. A mechanism of photochemical transformations of WO₃ films was suggested. This mechanism included the generation of electron-hole pairs, the recombination of part of nonequilibrium charge carriers, the formation of [eV_a²⁺e] centers, and the isolation of photolysis products.     

QUANTUM EFFICIENCY AND PHOTOSENSITIVITY OF THE RHODOPSIN ⇌ METARHODOPSIN CONVERSION IN CRAYFISH PHOTORECEPTORS

Photosensitivity (K_λ) of a visual pigment is the product of the molecular absorption coefficient (α_λ) and the quantum efficiency for photoconversion (γ). Among the invertebrates, many visual pigments are stable not only in the rhodopsin (R) conformation but also as the photoproduct, metarhodopsin (M), We here employ a method for determining the photosensitivities of the two stable pigments of a rhodopsin-metarhodopsin pair, using kinetic analysis of fluorescence from metarhodopsin combined with measurements of spectral absorption made before and after saturation at the isosbestic wavelength of the pigment pair. A curve fitting technique, in which a theoretical function is scaled for best fit to the measured absorption spectrum of the photosteady-state mixture, yields values for the photosensitivity of rhodopsin at λ._max, the ratio of quantum efficiencies for rhodopsin—metarhodopsin interconversion, and the fractional composition of the steady-state mixture. With knowledge of the molecular extinction coefficient, the absolute values of quantum efficiency can be calculated. For crayfish ( Orconectes, Procambarus ) rhodopsin, measured in isolated rhabdoms, K_max= 1.05 x 10^-16 cm² at 535 nm with >7λR→M0.69. These values are similar to the photosensitivity and quantum efficiency of bleaching of vertebrate rhodopsins in digitonin solution (Dartnall, 1972). For the metarhodopsin, K_max= 1.02 x 10^-16 cm² at 510 nm, and λ_M-R= 0.49.     

Radiation chemical investigations on aqueous solutions of C60(OH)18

The ultraviolet-visible absorption spectrum of C₆₀(OH)₁₈ in water showed an absorption band with λ_max = 215 nm and other characteristic absorption bands of C₆₀ are not observed. The singlet-singlet and triplet-triplet absorption bands are not observed in the 400–900 nm region. It has low reactivity with e_aq⁻ and formed an absorption band with λ_max = 580 nm. The hydroxyl radicals react with a bimolecular rate constant of 2.4×10⁹ dm³ mol⁻¹ s⁻¹ and showed an absorption band at 540 nm.     

ANALYTICAL MODEL OF PHOTOTHERAPY FOR NEONATAL JAUNDICE—I. THE INITIAL Z-E TRANSIENT

A simple model of phototherapy (PT) for neonatal jaundice is presented. Two coupled systems are considered: the "skin" (upper skin layers and vascular network where phototherapy light may penetrate) and the "body" (i.e. the "dark" ensemble of blood vessels and organs where circulation and metabolization of biliribin (BR) and its photoisomers occur).
The mathematics necessary to analyze the PT process is presented together with an appropriate optical model of skin based on a multilayered system to which the theory of radiation transfer in isotropically scattering and absorbing media is applied. A simple matrix formalism is introduced to determine analytically the light distribution in the multilayer skin. The results are. then, used to analyze the initial transient of PT, i.e. configurational isomerization up to photoequilibrium in the superficial skin layers under blue (450 nm) and green (500 nm) light irradiation within a time interval sufficiently short to neglect diffusion of pigments and formation of structural isomers of BR.
Strong coupling among the various BR layers due to the time variation of diffuse transmittance and reflectance is found, resulting in complicated time patterns with initial and final single-exponential behavior. The initial decay constant of BR concentration depends sensibly on the depth of the layer, while the final one is independent of it. As expected the amount of BR conversion at photoequilibrium is larger with blue than with green light. However, at sufficiently larger depths green light is more efficient than blue light to produce the initial conversion of ZZ to ZE isomers of BR.
The present analysis has some relevance for the understanding of the different efficiencies of coloured fluorescent lamps in clinical PT. The application of the complete model ("skin"+"body") requires the knowledge of the diffusion constants and absorption coefficients of pigments and of the optical parameters of deep cutaneous tissues.     

SPECTROPHOTOMETRIC IDENTITY OF THE ENERGY TRANSFER CHROMOPHORES IN RENILLA AND AEQUOREA GREEN-FLUORESCENT PROTEINS

Abstract— Spectral properties of guanidine-denaturated and pronase-digested green-fluorescent proteins (GFP) from two species of bioluminescent coelenterates have been investigated. Spectrophotometric titrations of Renilla and Aequorea GFP, following denaturation in 6 M guanidine HCl at elevated temperature, revealed identical absorption peaks in acid (383–384 nm) and in alkali (447–448 nm) and a single isosbestic point in the visible region at 405 nm. Both proteins exhibited a spectrophotometric pK. of 8.1 in guanidine -HCl. Pronase digestion of the heat-denaturated GFP's generated a methanol-soluble blue-fluorescent peptide with identical fluorescence emission spectra (λ_max= 430 nm, uncorrected; φ_f1= 0.003) for both coelenterate species. These data suggest that the large absorption differences between native Renilla and Aequorea GFP molecules result from unique protein environments imported to a common chromophore.     

HALORHODOPSIN and PHOTOSENSORY BEHAVIOR IN Halobacterium halobium MUTANT STRAIN L-33

Halobacterium halobium , strain L-33, which is deficient in bacteriorhodopsin (BR) but synthesizes increased amounts of halorhodopsin (HR), shows behavioral responses upon changes in fluence rate with visible light or with UV light. The observations support the earlier report (Schimz et al. , 1982). that BR is not essential for photosensing in H. halobium. In the UV-range, changes in light intensity elicit the maximal response at λ= 370 nm. In the visible range, changes in light intensity show the maximal response at Δ= 565 nm and a secondary peak at Δ= 590 nm. The latter corresponds to the absorption maximum of HR (Δ_max= 588 nm). This light-energy converting retinal pigment of H. halobium thus appears to contribute to photosensory behavior.     

Diffuse Reflectance Spectroscopy as a Reliable Means of Comparing Ultraviolet Radiation‐induced Erythema in Extreme Skin Colors

Erythema is widely considered an indicator of skin cancer susceptibility, but assessments are challenging in black skin because melanin can mask erythema under traditional visual and advanced objective Commission Internationale de l'Eclairage (CIE) L *a *b * assessments. Using spectral measurements (400–700 nm) from a spectrophotometer, an algorithm was developed to measure erythema in white Caucasians (n = 9) and black West Africans (n = 11) 19–24 h postsolar simulated radiation (SSR) exposures to the volar forearm. The derived spectrum achieved showed a strong maximum peak for hemoglobin at 580 nm and a linear slope between 650 and 700 nm for melanin absorption, as reported by other authors. Absorption by hemoglobin at 580 nm was used as a proxy for erythema, and melanin was quantified between 650 and 700 nm. Our algorithm corrected the erythema measurements for stray specular (mirror‐like) reflection and the melanin‐masking effect. A linear relationship between SSR exposure and erythema was evident (p < 0.0001 for white and black skin), and white skin is 8.4 times more responsive to SSR compared to black skin. The prediction of ultraviolet radiation sensitivity is vital in both clinical and investigative dermatology especially in the determination of starting phototherapy doses. Our methodology allows for the accurate assessment of erythema independent of constitutive pigmentation.     

PHOTOBINDING OF CHLORPROMAZINE AND ITS SULFOXIDE IN VITRO AND IN VIVO

Abstract— Photosensitivity as observed after chlorpromazine (CPZ) treatment is enhanced in the UVA- rather than the UVB region, whereas CPZ has its absorption maximum at 305 nm. This long wavelength sensitivity has sometimes been ascribed to CPZ-sulfoxide (CPZSO) which has an absorption maximum at 340 nm. We compared the photobinding properties of CPZSO and CPZ under both in vitro and in vivo conditions.
With 310 and 370 nm lamps CPZSO absorbs twice as much light as CPZ but still binds less efficiently to HSA in vitro. At wavelengths longer than 380 nm CPZSO does not absorb nor photobind to HSA (420 nm lamps) in contrast to CPZ.
In vivo the bioavailability of CPZ and CPZSO in ears, eyes and skin of the back of Wistar rats is comparable, yet irradiation with 370 nm light caused more CPZ-photobinding in these tissues. Chlorpromazine binds relatively more compared to CPZSO, to constituents of deeper lying tissues (dermis). This corresponds with the observation that both the ratio of in vitro CPZ photobinding to CPZSO photobinding, and the penetrating ability of light in the skin increase with wavelength.
In the eyes, where the lens efficiently filters out light with wavelengths shorter than 370 nm, no CPZSO photobinding was observed, in contrast to CPZ; this also corresponds with the in vitro experiments. Therefore it seems more likely that the observed wavelength maximum in the photosensitivity action spectrum after CPZ treatment should be attributed to the non-sulfoxidated drug rather than to the sulfoxidated compound.     

BIOPHYSICAL AND BIOCHEMICAL ASPECTS OF PHENGODID (RAILROAD-WORM) BIOLUMINESCENCE

In studies of the bioluminescence of 11 species of phengodid collected in central and southeast Brazil, we have found that: (1) their lateral lanterns emit light in the yellow-green region (λ_max= 540–580 nm) and the head lantern color is shifted to the red region ( λ_max= 565–620 nm), (2) the luciferins of both types of lanterns are identical to that of lampyrids and elaterids and (3) the luciferase physicochemical properties are also similar to those of lampyrids and elaterids (optimum pH ca 8.1; K_m(ATP) = 260–370 μM , Kμ(luciferin) = 170–400 μM; molecular weight ca 60 kDa; apparent activation energy of in vitro bioluminescence ca 58 kJ/mol). Thus the bioluminescence system of phengodids appears to be essentially the same as that of lampyrids and elaterids. The different bioluminescence colors of the lanterns of Phrixothrix species (λ_head= 600–620 nm; λ_lateral= 535–565 nm) and other phengodid species are probably elicited by the presence of luciferase isoenzymes, as occurs in the case of elaterid prothoracic and abdominal lanterns.     

CONFIGURATIONAL PHOTOISOMERIZATION OF BILIRUBIN IN VITRO—I. QUENCHING OF Z→E ISOMERIZATION BY TWO-WAVELENGTH IRRADIATION

Abstract A simple rate-equation theory of configurational photoisomerization of bilirubin (BR) is presented and used to discuss the Z→E photoisomerization of BR in HSA solutions irradiated with laser light in the blue-green spectral region. The analytical expression of Z, E -BR:HSA (PBR) concentration at photoequilibrium (PE) is used to derive the PBR concentration from the spectrophotometric data on absorbance difference spectra. The PBR values are in good agreement with those recently reported in the case of 10 nm-bandwidth filtered light. Both sets of data give a best-fit value of the BR, PBR quantum yield ratio equal to 2.3 in the case of BR:HSA ratio equal to 1:2.
Successive irradiation of BR/HSA solutions with different wavelengths (457/488/457 nm) provides further support for the reversibility of the photoreaction. However, differences between the intermediate PE values have been found, and may reveal departures from the simple Z,Z→Z,E isomerization in BR/HSA solutions.
The possibility of controlling the formation of Z, E -BR/HSA has been demonstrated using simultaneous irradiation with blue (457 nm) and green (514 nm) laser beams. The two-wavelength scheme takes advantage of the reversibility of the BRIPBR reaction, and of the differences between BR/PBR absorption spectra and quantum yields. Efficient quenching of PE PBR is achieved when green substantially exceeds (10–40 times) blue light intensity. This experiment may simulate what actually occurs during clinical phototherapy with white and green fluorescent lamps, where the quenching of PBR at the formation sites may prevent diffusion of this slowly excreted isomer and, at the same time, increase the formation of the more rapidly excretable structural, non-reversible isomers, such as lumirubin.     

PHOTOACTIVABLE FLUOROPHORES FOR THE MEASUREMENT OF FLUENCE IN TURBID MEDIA

Knowledge of the fluence distribution in biological tissue is essential for applications of lasers and light in medicine. A method using a photoactivable fluorophore as a chemical actinometer is presented to investigate the fluence (J/cm²) distribution in tissue-simulating phantoms. Such a chemical actinometer provides high spatial resolution (≤20 μm) while minimizing the disturbance of the fluence distribution. The actinometer substance, nonfluorescent in its native state, is incorporated into an acrylamide gel. Upon absorption of 351 nm radiation (λ_act), the actinometer substance becomes a Ruorophorc, which is excited at λ_ex≤ 485 nm. Thus the spatial distribution of the emitted fluorescence (λ_em≤ 515 nm) in the actinometer represents the fluence distribution of the activating radiation. Using histological techniques, 20 μm sections are cut from gel-like optical phantoms containing the actinometric substance. The fluorescence intensity in the section is recorded under a standard fluorescence microscope equipped with a sensitive video camera. To simulate different biological tissues, the scattering and absorption properties of the gel phantoms arc varicd over a wide range. The experimentally obtained fluence distributions are compared with theoretical models of light distribution in turbid media.     

Characterization of Photodegradation of Meta-tetra (Hydroxyphenyl)chlorin (mTHPC) in Solution: Biological Consequences in Human Tumor Cells

The photobleaching of meta -tetra(hydroxyphenyl)chlorin m THPC) (irradiation wavelength 413 nm) in protein-containing solution was evaluated by decay in absorbance in Soret band and in fluorescence (λ_exc= 423 nm, λ_em= 655 nm). Light exposure resulted in a decrease in absorption throughout the spectrum and simultaneous appearance of new absorption bands in the spectral region 325–450 nm. The rate of m THPC photodegradation, followed by decay in absorbance, was 15-fold lower than that observed in fluorescence. This fact reflects the photobleaching of presumably monomeric, fluorescing species of m THPC. In order to determine the consequences of photobleaching of fluorescing m THPC material on cellular uptake and photocytotoxicity, human HT29 colon adenocarcinoma cells were incubated with photobleached m THPC during 5 h with or without following irradiation with the fixed fluence. Surprisingly, but up to the time when the fluorescence decreased by 50%, only a slight decrease in photocytotoxicity was detected. Either aggregated forms that have been taken up undergo intracellular monomerization (but we did not observe increase in fluorescence in living cells) or the photodynamic activity is mostly due to aggregates. The discrepancy of m -THPC-photodynamic therapy (PDT) effect and fluorescence measurements may suggest that aggregated m -THPC plays an important role in m THPC-PDT.