QUANTUM YIELDS FOR THE CYCLIZATION AND CONFIGURATIONAL ISOMERIZATION OF 4E, 15Z-BILIRUBIN

Extraction of a solution of bilirubin configurational isomers in chloroform with an aqueous solution of human serum albumin was found to remove selectively the 4Z,15E-isomer. This phenomenon was used to develop a method for the purification of the 4E,15Z-isomer of bilirubin. The quantum yield for the cyclization and configurational isomerization of the 4E,15Z-isomer bound to a molar excess of human serum albumin was measured at 450 and 510 nm. The quantum yield for cyclization to form lumirubin was 0.12 and 0.19 at 450 and 510 nm, respectively. The quantum yield for configurational isomerization to form 4Z,15Z-bilirubin was 0.03 and 0.05 at 450 and 510 nm. An analysis of previously published data on the quantum yield for the formation of lumirubin from 4Z, 15Z-bilirubin bound to human serum albumin suggests that all of the formation of lumirubin may occur via consecutive photochemical processes with the 4E,15Z-isomer as an intermediate.     

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.     

Accurate and sensitive high-performance liquid chromatographic method for geometrical and structural photoisomers of bilirubin IX alpha using the relative molar absorptivity values.

It has been reported that considerable differences exist between the relative molar absorptivity values of the geometrical and structural photoisomers of bilirubin. We have devised an accurate HPLC method for photoisomer quantification based on the following principle: the sum of both the integrated peak areas corrected by each factor for each photoisomer, and the integrated peak area of unchanged (ZZ)-bilirubin [(ZZ)-B] after an anaerobic photoirradiation, should be constant and equal to the integrated peak area of initial (ZZ)-bilirubin [(ZZ)-Bi] before photoirradiation. On this basis, the following equation can be used to determine each factor. [equation: see text] alpha, beta, gamma and delta represent the factors used to correct the integrated peak areas of individual bilirubin photoisomers, and they are arranged in the order of the formula. It was demonstrated that the relative 455 nm molar absorptivity values for (ZZ)-bilirubin and all its geometrical and structural photoisomers, i.e., (ZZ)-bilirubin, (ZE)-bilirubin (EZ)-bilirubin, (EZ)-cyclobilirubin (= lumirubin) and (EE)-cyclobilirubin in the HPLC eluent, are, respectively, 1.0, 0.81 (= alpha), 0.54 (= beta), 0.47 (= gamma) and 0.39 (= delta).     

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.     

WAVELENGTH-DEPENDENT QUANTUM YIELD FOR Z →E ISOMERIZATION OF BILIRUBIN COMPLEXED WITH HUMAN SERUM ALBUMIN

The quantum yield, phi ZE, for configurational photoisomerization (4Z,15Z----4Z,15E) of bilirubin bound non-covalently to human serum albumin was determined (at 23 +/- 2 degrees C) by laser excitation and chromatographic analysis of products. Values obtained for photoexcitation at 465 nm were about one-half those previously reported. The quantum yield was dependent on excitation wavelength, decreasing from a value of 0.109 +/- 0.010 for excitation at 457.9 nm to a value of 0.054 +/- 0.005 for excitation at 514.5 nm. The wavelength dependence is consistent with rapid transfer of excitation energy between the two non-identical pyrromethenone chromophores of bilirubin in the singlet excited state. Since the quantum yields for photoisomerization and luminescence of bilirubin bound to serum albumin at room temperature are both low, internal conversion processes, rather than Z----E configurational isomerizations, are probably the major pathways for deactivation of photo-excited bilirubin.     

Long Chain Fatty Acid Affects Excited State Branching in Bilirubin-Human Serum Protein Complex?

After binding to human serum albumin, bilirubin could undergo photo-isomerization and photo-induced cyclization process. The latter process would result the formation of a product, named as lumirubin. These photo induced behaviors are the fundamental of clinical therapy for neonatal jaundice. Previous studies have reported that the addition of long chain fatty acids is beneficial to the generation of lumirubin, yet no kinetic study has revealed the mechanism behind. In this study, how palmitic acid affects the photochemical reaction process of bilirubin in Human serum albumin (HSA) is studied by using femtosecond transient absorption and fluorescence up-conversion techniques. With the addition of palmitic acid, the excited population of bilirubin prefers to return to its hot ground state (S₀) through a 4 ps decay channel rather than the intrinsic ultrafast decay pathways (< 1 ps). This effect prompts the Z-Z to E-Z isomerization at the S$_0$ state and then further increases the production yield of lumirubin. This is the first time to characterize the promoting effect of long chain fatty acid in the process of phototherapy with femtosecond time resolution spectroscopy and the results can provide useful information to benefit the relevant clinical study.     

QUANTUM YIELD and EQUILIBRIUM POSITION OF THE CONFIGURATIONAL PHOTOISOMERIZATION OF BILIRUBIN BOUND TO HUMAN SERUM ALBUMIN*

Abstract— At low fluence rates it is possible to observe a photoequilibrium between Z,Z-bilirubin(BR) and its E isomers(collectively called PBR) bound to human serum albumin(HSA).For excitation centered at 465 nm, the fraction of PBR/HSA in the photoequilibrium mixture was observed to be 0.22 + 0.02. The quantum yield for conversion of BR/HSA to PBR/HSA was found to be 0.20 0.02. The equality of the quantum yield value and the fraction of PBR/HSA in the photoequilibrium mixture is consistent with a simple mechanism for the photoisomerization in which optically excited singlet states of BR and PBR convert rapidly and with virtually total efficiency to a common excited intermediate with twisted geometry that subsequently decays to BR and PBR in the ratio 4:1, respectively. Quantum yields for other photoprocesses of BR bound to HSA are much lower than that for configur-ational isomerization. The central role suggested for configurational isomerization in the phototherapy for neonatal hyperbilirubinemia is supported by these observations.     

A new photoisomerization of bilirubin

Photoirradiation of solutions of natural (4Z,15Z)-bilirubin-IX alpha in chloroform with approximately 366 nm UV light leads rapidly to a new photoisomer that has been characterized by NMR spectroscopy and by its (ground-state reaction) adducts with methanol and other protic nucleophiles. Unlike the previously described Z-->E geometric isomerization important in phototherapy of neonatal jaundice, the new photoisomerization involves regiospecific photoautomerization to afford 2-ethenyl-18-ethylidene- 1,10,19,22,24-pentahydro-2,7,13,17-tetramethyl-1,19- dioxo-21H-biline-8,12-dipropanoic acid.     

SKIN OPTICS AND PHOTOTHERAPY OF JAUNDICE

Abstract The Kubelka–Munk theory of radiation transfer is applied to determine the influence of skin optical losses on the efficiency of phototherapy of jaundice. Using a multi-layer model of the skin the photon absorption rate of bilirubin molecules is calculated for spectrally Gaussian light sources and fluorescent lamps used in phototherapy. Light absorption and scattering processes in the skin layers shift the optimum value of the peak excitation wavelength from λ= 453 nm (absorption maximum of bilirubin in vitro ) to λ= 480 nm. This suggests the clinical investigation of narrow-spectrum lamps emitting in the blue-green region of the spectrum.     

Photochromic fused-naphthopyrans without residual color

A series of new photochromic fused-naphthopyrans with an alkyl bridge between the pyran ring and the naphthalenic core was synthesized in several steps from 4-(bromomethyl)benzocoumarin. The presence of the alkyl bridge in these new fused-naphthopyrans prevents the formation of one long-lived photoisomer and therefore has a dramatic effect on their photochromic properties: UV irradiation of common naphthopyrans gives rise to two isomeric colored photoisomers, one of which fades very slowly and is responsible for a persistent residual color. UV excitation of these new uncolored fused-naphthopyrans leads to the formation of only one colored photoisomer that fades completely to the uncolored state in few seconds/minutes following a monoexponential decay law, thus avoiding the problem of the residual coloration typically observed with naphthopyrans.     

Characterization of the photoisomers from cis- and trans-chlordanes, trans-nonachlor and heptachlor epoxide

Irradiation of acetone solutions of trans-chlordane (4a) and trans-nonachlor (4b) with UV light produces new half-cage photoisomers (5a or 6a and 5b or 6b, respectively) with bridging that differs from that of the photoisomers (2 and 8a) obtained from cis-chlordane (1b) and heptachlor epoxide (7). A new photoisomer (10) obtained from heptachlor epoxide is transformed into photoisomer 8a on further irradiation. Detailed PMR and ¹³C-NMR studies establish the structures of the new photoisomers and permit a decision between alternative formulations for the structures of the half-cage photoisomers of heptachlor epoxide and cis-chlordane,     

NMR kinetic investigations of the photochemical and thermal reactions of a photochromic chromene

The photochromic behavior of 2,2-di(4-fluorophenyl)-6-methoxy-2H-1-chromene has been investigated by 19F NMR spectroscopy. Photocoloration under UV irradiation at low temperature led to the formation of three interconverting photoisomers including two merocyanines and an unprecedented allenyl-phenol isomer. Photobleaching with visible light, which was known to lead to reversion to the initial closed chromene, was shown to increase allenyl-phenol concentration. Thermal relaxation of the preirradiated system was also studied at various temperatures. In each case (UV and visible irradiations, thermal isomerization), the kinetics of each of the four species was monitored. Numerical analysis of concentration vs time profiles enabled us to unequivocally establish the global mechanism occurring in each of the experimental conditions and to interpret the specific reactivity of each photoisomer. It has been shown that, among the 12 possible isomerization processes, only some paths were active. For the first time, it has been possible to determine their corresponding thermal activation parameters and photochemical quantum yield ratios.     

The Irreversible Photochemistry of Bianthrone

A 4a,4b-dihydrophenanthrene-type cyclic photoisomer, the C isomer, is the major primary photoproduct of bianthrone in protic and aprotic polar solvents, and undergoes solvent-dependent secondary reactions, including the formation of dihydrohelianthrone in protic solvents. The C isomer was shown to be formed through the singlet excited state while the B isomer is formed via the triplet manifold.     

Toward an amphiphilic bilirubin: the crystal structure of a bilirubin e-isomer

A new bilirubinoid analog (1) with two methoxy beta-substituents on the lactam ring of each dipyrrinone was synthesized and examined spectroscopically. It is more soluble in CH3OH and CHCl3 than bilirubin, which is insoluble in CH3OH but soluble in CHCl3. The solubility of 1 is approximately 10 microg/mL in CH3OH (vs < or =1 microg/mL for bilirubin) and approximately 3 mg/mL in CHCl3 (vs approximately 0.6 mg/mL for bilirubin). Vapor pressure osmometry indicates that 1, like bilirubin, is monomeric in CHCl3, and NMR studies show that the most stable structure has the syn-4Z,syn-15Z configuration, with the pigment's dipyrrinones engaged in intramolecular hydrogen bonding to the propionic acid carboxyl groups. And, like bilirubin, Z,Z-1 adopts a conformation that is bent in the middle into a ridge-tile shape. For the first time, a crystal structure of a bilirubin E-isomer has been obtained. Crystallization of 1 under dim room lighting gave an X-ray quality crystal of the anti-4E,syn-15Z-(photo) isomer, in which only the Z-dipyrrinone half is engaged in intramolecular hydrogen bonding to a propionic acid. Hydrogen bonding is nearly completely disengaged in the E-dipyrrinone half; yet, the ridge-tile conformation persists.     

(EZ)-Cyclobilirubin formation from bilirubin in complex with serum albumin derived from various species

We determined the specificity of photochemical changes of bilirubin in complex with serum albumin from various species. There were no general trends in the configurational photoisomerizations of (ZE)-bilirubin/(ZZ)-bilirubin and (EZ)-bilirubin/(ZZ)-bilirubin associated with the albumins from various species as compared to those associated with human serum albumin. The absorbance spectra of bilirubin in complex with albumins from various species differed, indicating that the three-dimensional structures of (ZZ)-bilirubin bound to the various serum albumins, which are substrates of (ZE)- and (EZ)-bilirubin, differ among species. The rates of conversion of the (EZ)-bilirubin isomer into the structural cyclobilirubin isomer were similar for the albumins of chicken, rat, rabbit, dog, bovine, and pig, and were significantly slower than the rate for human serum albumin. This suggests that the three-domain human albumin has evolved to allow ready conversion of (EZ)-bilirubin to (EZ)-cyclobilirubin. Cyclobilirubin formation in a bilirubin-alpha-fetoprotein solution was much lower than that in a bilirubin–human serum albumin solution. It is believed that the ability of human serum albumin to facilitate the photochemical change of bilirubin was evolutionarily selected in response to neonatal jaundice in humans.     

Bilirubin- and light induced cell death in a murine lymphoma cell line

Cells from the mouse lymphoma cell line L5178Y-R were exposed to blue light from phototherapy lamps in the presence of solutions of 160 microM bilirubin supplemented with serum albumin. HPLC analysis showed that the bilirubin solution was photooxidised as a function of increasing light dose. The cells were stained with trypan blue to score necrosis, and apoptosis was assayed by the terminal deoxynucleotide transferase assay (TdT) or by studying the nuclear structure in cells stained with propidium iodide. A rapidly developing apoptosis was observed after light doses killing 60-80% of the cells as judged from the trypan blue exclusion test. The fraction of apoptotic cells was smaller than the fraction of necrotic cells. Exposure of the cells to fractions of light at a high dose rate was compared to the effect of the same total dose at a lower dose rate given as a single fraction. No large differences were found, however, there was a tendency of a higher degree of necrosis as well as apoptosis in the cells receiving the light in fractions at a high dose rate.     

Alkoxyamine-derived formamidines: configurational control and molecular folding

N,N'-Disubstituted formamidines, and amidines in general, have very rich configurational, conformational, and tautomeric diversities. As part of an effort to incorporate alkoxyamine-derived formamidine units into foldamers, the first evidence for the isolation of the up-to-now unknown E isomer, the conditions for its exclusive formation, its stability and self-assembly properties, and its configurational isomerization to its much more common Z counterpart are reported. Considering the distinctly different H-bonding patterns displayed by both E and Z isomers, such configurational control may find applications in self-assembly, molecular recognition, and biomimetic systems.     

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.     

New aspects of diphenylbutadiene photochemistry. Regiospecific hula-twist photoisomerization

In EPA glass at liquid nitrogen temperature, the E,E isomer of diphenylbutadiene (DPB) was photostable, while both the Z,E and Z,Z isomers underwent selective HT isomerization at center 1 giving the stable conformer of the double-bond isomerized trans product. That HT-1 was involved rather than the OBF process was shown by results of o,o'-dimethyl-DPB. Formation of unstable trans product corresponded to simultaneous configurational and conformational isomerization. The regioselectivity was found not sensitive to a substituent effect, as shown by the similar reactivity in p,p'- or o,o'-bistrifluoromethyl-DPB.