Kinetic study of the photochemical changes of (ZZ)-bilirubin IX alpha bound to human serum albumin. Demonstration of (EZ)-bilirubin IX alpha as an intermediate in photochemical changes from (ZZ)-bilirubin IX alpha to (EZ)-cyclobilirubin IX alpha.

The present study was performed to elucidate why the photochemical reaction of (ZZ)-bilirubin bound to human serum albumin is singularly selective, and only one of the two (EZ)- and (ZE)-bilirubins, the (ZE)-isomer, is produced. In a kinetic study of the photochemical reaction in vitro, the sum of the relative rate constants of photochemical transformation of (EZ)-bilirubin into both (EZ)-cyclobilirubin and (ZZ)-bilirubin, with a significant preference for the former, was proved to be considerably larger than that of the transformation of (ZZ)-bilirubin into (EZ)-bilirubin. Therefore only one of the geometrical isomers, namely (ZE)-bilirubin, is apparently formed. It was concluded that (EZ)-bilirubin photochemically undergoes (EZ)-cyclization, i.e. structural photoisomerization, while bound to its high-affinity site on human serum albumin, and is an intermediate in the transformation of (ZZ)-bilirubin into (EZ)-cyclobilirubin.     

Comparison of kinetic study of the photochemical changes of (ZZ)-bilirubin IX alpha bound to human serum albumin with that bound to rat serum albumin.

It has been stated by McDonagh, Palma & Lightner [(1982) J. Am. Chem. Soc. 104, 6867-6871] that complexing of bilirubin with serum albumin has a marked species-dependent influence on bilirubin photoisomerization in vitro and in vivo. Therefore the kinetics for the quantitatively important reaction: (Formula: see text) of the photochemical interconversion between bilirubin and its photoisomers bound to human or rat serum albumin in aqueous solution, assayed by h.p.l.c., was used to elucidate the observed species-dependent difference. The relative rate constants for bilirubin bound to human serum albumin, except for k4, the rate of interconversion from (ZZ)-bilirubin into (EZ)-bilirubin, proved to be considerably larger than those for bilirubin bound to rat serum albumin. In accordance with these rate constants, the formation of photoisomers of bilirubin bound to human serum albumin, except for (EZ)-bilirubin, is very rapid and much greater than that for bilirubin bound to rat serum albumin.     

Metabolism of bilirubin and its photoisomers in newborn infants during phototherapy

Bilirubin and its photoisomers in the biological fluids of a hyperbilirubinaemic newborn infant before and during phototherapy were analyzed by a recently improved HPLC method. In the serum, the percentages of (EZ)- and (ZE)-bilirubin in the total bilirubin concentration before phototherapy were approximately 10% and on average increased over 1.5-fold at 2 h after initiation of phototherapy. The percentage of the (EZ)-cyclobilirubin in the serum bilirubin was under 1%. In the bile, the mean concentration of (ZZ)-bilirubin, derived mainly from (ZE)-bilirubin, nearly tripled during phototherapy. The (EZ)-cyclobilirubin concentration in the bile was very low before phototherapy, increased nearly ten-fold at 3 h after initiation of phototherapy, and was 5- to 6-fold as high as that of (ZZ)-bilirubin. In the urine, upon exposure to light, the urinary concentration of (EZ)-cyclobilirubin is apparently equivalent to half of the biliary concentration of (ZZ)-bilirubin and one-fifth of that of (EZ)-cyclobilirubin. It was concluded that during phototherapy of neonatal hyperbilirubinaemia the structural photoisomer [(EZ)-cyclobilirubin] predominates considerably over the geometric photoisomer [(ZE)-bilirubin].     

Biliary and urinary excretion rates and serum concentration changes of four bilirubin photoproducts in Gunn rats during total darkness and low or high illumination.

On cycled exposure of Gunn rats to total darkness and low and high illumination, biliary excretion rates of (EZ)- and (ZE)-bilirubin and (EZ)-cyclobilirubin increased up to approx. 10-fold from the mean basal values of 1.2 and 0.2 microgram/h to the mean maximum values of 25.2 and 4.2 micrograms/h respectively, and at the same time those of (EE)-bilirubin and (EE)-cyclobilirubin also increased, but at very much lower rates than those of the first-mentioned two. During the low illumination only (EZ)- and (ZE)-bilirubin and (EZ)-cyclobilirubin appeared in the urine; during the high illumination (EE)-bilirubin and (EE)-cyclobilirubin also appeared, showing a similar excretion pattern to that observed in the bile, but the total urinary excretion rates were lower than the total biliary excretion rates. The serum bilirubin concentrations fell gradually to lower values, accompanied by an increment in (EZ)- and (ZE)-bilirubin, but (EZ)-cyclobilirubin was not detected. It is concluded that during phototherapy the predominant pathway for the removal of bilirubin from the body in the Gunn rat is by biliary excretion of the geometric photoisomers (EZ)- and (ZE)-bilirubin, derived from Z----E isomerization, and the structural photoisomer (EZ)-cyclobilirubin, formed from intramolecular endo-vinyl cyclization.     

Structure and thermal interconversion of cyclobilirubin IX alpha.

One of the two main photoproducts in bilirubin metabolism during phototherapy in neonatal hyperbilirubinaemia is (EZ)-cyclobilirubin. However, it has not yet been possible to come to a final conclusion as to its chemical structure, despite the fact that much effort has been expended on the problem. The present paper demonstrates that (EZ)-cyclobilirubin is formed by the intramolecular cyclization of the C-3-vinyl group with the position at C-7 rather than at C-6, without delta-lactone-ring formation. The evidence comes from 13C-n.m.r. spectra, which indicate that an oxygen-bound quaternary carbon atom is not present, and from 1H-n.m.r. spectra, which indicate that the orientation of the methyl group at C-2 is equatorial; these findings are supported by mass spectra. The existence of both an epimeric relationship at C-7 between (EE)- and (EZ)-cyclobilirubins A and B and of steric isomers of the hydrogen atom and methyl group at C-2 is supported by the fact that the methyl-group protons at C-2 and C-7 are observed as a paired signal in 1H-n.m.r. spectra, and that new signals at C-7, C-2 and C-3 beta appear in 13C-n.m.r. spectra, that mass spectra of (EZ)-cyclobilirubins A and B are extremely similar and that, furthermore, thermal interconversion between (EE)- and (EZ)-cyclobilirubins A and B is observed.     

The effect of bilirubin photoisomers on unbound-bilirubin concentrations estimated by the peroxidase method.

Unbound bilirubin is oxidized to nearly colourless substances in the presence of H2O2 or ethyl hydroperoxide and horseradish peroxidase. To predict the risk of kernicterus (degenerated yellow pigmentation of nerve cells), this principle has been widely utilized for estimating the concentration of unbound bilirubin in hyperbilirubinaemic serum. However, the serum contains polar geometric photoisomers of bilirubin. Therefore, to clarify the effect of bilirubin photoisomer concentrations on unbound-bilirubin concentration, the concentration of bilirubin and its photoisomer and of unbound bilirubin in samples obtained from experiments in vivo and in vitro were simultaneously and individually estimated by h.p.l.c. and the peroxidase method. During photoirradiation, both in vivo and in vitro, the serum polar (ZE)-bilirubin IX alpha concentration increased remarkably, but unbound-bilirubin values were not affected at all. However, during experiments in vitro, unbound bilirubin concentrations increased only when concentrations of the rather polar (EZ)- and (EE)-cyclobilirubin IX alpha increased considerably in a human serum albumin-bilirubin solution irradiated with blue light. Thus it is concluded that unbound-bilirubin concentrations, and consequently the initial rate of the peroxidase reaction, is not accelerated by the increase in either (ZE)-bilirubin or (EZ)-cyclobilirubin concentration within the clinically observed range.     

Effect of the binding of bilirubin to either the first class or the second class of binding sites of the human serum albumin molecule on its photochemical reaction.

The kinetics of the photochemical changes of bilirubin were studied at a constant concentration of bilirubin bound either to the first class or to the second class of binding sites of the human serum albumin molecule. The more the bilirubin binds to the first class of binding sites in the human serum albumin molecule, the more readily geometric photoequilibrium to give (ZE)-bilirubin takes place. The more the bilirubin binds to the second class of binding sites or allosterically transformed binding sites induced by added SDS, the more readily structural photoisomerization, i.e. the formation of (EZ)-cyclobilirubin, takes place. When the serum bilirubin concentration is at low, safe, values bilirubin binds exclusively to the first class of binding sites and serves as an antioxidant [Onishi, Yamakawa & Ogawa (1971) Perinatology 1, 373-379]; at these concentrations human serum albumin protects bilirubin from irreversible photodegradation by only allowing readily reversible geometric photoisomerization. As the serum bilirubin concentration increases to high, and potentially dangerous, values, bilirubin binds to the second class of binding sites, and under these conditions human serum albumin seems to promote the photocyclization of bilirubin. During irradiation human serum albumin seems to act by retaining low, useful, concentrations of bilirubin while facilitating irreversible photoisomerization of excess bilirubin.     

Possible antagonism of (Z)‐2‐hexenyl (E)‐3‐hexenoate against the attractiveness of (E)‐2‐hexenyl (Z)‐3‐hexenoate to Ooencyrtus nezarae (Hymenoptera: Encyrtidae)

Ooencyrtus nezarae (Hymenoptera: Encyrtidae) is an egg parasitoid of bean bug Riptortus pedestris (Hemiptera: Alydidae) which is a major pest of beans. Females of O. nezarae are attracted to (E)‐2‐hexenyl (Z)‐3‐hexenoate (EZ), one of the components of aggregation pheromone of R. pedestris. Effects of three isomers (ZE, EE and ZZ) of EZ on the attractiveness of O. nezarae were tested using electroantennography (EAG) and field bioassays. EAG analyses revealed that the response of O. nezarae to ZE was significantly higher than those to air, hexane and two other isomers, even though the response was lower than that to EZ. ZE affected the attractiveness of EZ dose‐dependently in the field. Addition of ZE (100 mg) to EZ (10 mg) caused a significant reduction in the catches of O. nezarae females. Single or binary addition of two other isomers (EE and ZZ) to EZ could not decrease or increase significantly the number of O. nezarae catches of EZ. Even though addition of ZZ (10, 50 or 100 mg) to EZ (10 mg) caused dose‐dependent reduction in the number of O. nezarae female catches, the reductions were not significantly different from that of EZ. EZ and its three isomers were not attractive to O. nezarae males at all.     

Kinetics of biliary excretion of the main two bilirubin photoproducts after injection into Gunn rats.

The kinetics of biliary excretion of the main two photoproducts after injection into Gunn rats were examined. The photoproducts that are obtained from experiments in vitro consist of unknown pigment, photobilirubin IXa and a small amount of (ZZ)-bilirubin IXa. It was confirmed previously that the first two photoproducts are identical with the main two photoproducts obtained in vivo. In experiments on four animals, the average of total biliary recoveries of unknown pigment was 81.4%, and that of photobilirubin IXa in the bile estimated by the Sigma-minus method was 29.8 min and that for unknown pigment was 4.3 min. The rate of thermal reversion of photobilirubin IXa to (ZZ)-bilirubin IXa in the bile at 37 degrees C was very rapid, i.e. its half-life was 6.2 min.     

Significance of the endo-vinyl group of bilirubin in photochemical reactions.

In photochemical experiments on bilirubin III alpha (no endo-vinyl group), IX alpha (one endo-vinyl group) and XIII alpha (two endo-vinyl groups) and in the photochemical, thermal and catalytical reversion of their photoproducts under anaerobic conditions, much more instability and complexity of photoproducts of bilirubin XIII alpha were observed than for those of bilirubin IX alpha or III alpha. On the basis of present and previous results of photochemical experiments in vitro and the fact that large amounts of (EZ)-cyclobilirubin IX alpha appear in the bile during phototherapy of neonatal hyperbilirubinaemia [Onishi, Kawade, Itoh, Isobe & Sugiyama (1980) Biochem. J. 190, 527-532], it is concluded that the endo-vinyl group plays a crucial role in the photochemical reaction of bilirubin IX alpha. On reversed-phase high-pressure liquid chromatography of photoisomers, it was found that the retention times of geometric isomers and E-cyclized structural isomers were shortened compared with those of Z-isomer and E-isomer, respectively, as precursor substances.     

Bilirubin photoconversion induced by monochromatic laser radiation. Comparison between aerobic and anaerobic experiments in vitro.

Structural and geometric photoisomerization of bilirubin bound to human serum albumin was investigated. Solutions were irradiated with monochromatic light emitted by an Ar ion laser, the 457.9, 488.0 and 514.5 nm wavelengths being selected. Photoproducts were separated and analysed by h.p.l.c. Visible-absorption spectra of pure ZZ-bilirubin, ZE-bilirubin and lumirubin in the eluent were registered in the 350-550 nm region by collecting single fractions by h.p.l.c. Wavelength-dependence of bilirubin photoconversion was studied within photoequilibrium and up to a large decrement of the total concentration. Experiments were performed in aerobic and anaerobic conditions in order to assess the contribution of the photo-oxidation to the overall process. The presence of O2 was found to increase the rate of bilirubin degradation and unexpectedly to favour lumirubin production. The ability of 514.5 nm irradiation to induce bilirubin cyclization was definitively confirmed.     

Rapid spectral scan and stopped-flow studies of carbon monoxide binding to bovine adrenocortical cytochrome P-450scc

Carbon monoxide binding with both cholesterol-free (low-spin) and cholesterol-bound (high-spin) reduced forms of purified cytochrome P-450scc has been investigated by rapid-scan and stopped-flow spectrometry. CO binding occurs within 150 ms at 25 degrees C for both forms of P-450scc, with a typical absorption maximum at 450 nm. Isosbestic points occur at the following wavelengths: between reduced-CO and reduced cholesterol-free P-450scc at 434 and 471 nm; between reduced-CO and reduced cholesterol-bound P-450scc at 433 and 469 nm. Both the 'on' (k1) and 'off' rate constants (k-1) are found to be independent of pH between pH 5 and 9. The mean values of k1 for cholesterol-free (1.8 +/- 0.2) X 10(5) M-1 X s-1) and cholesterol-bound [1.9 +/- 0.1) X 10(5) M-1 X s-1) P-450scc are almost identical, while the mean value of k-1 for the former [2.3 +/- 0.3) X 10 s-1) is about double that of the latter [1.2 +/- 0.1) X 10 s-1). This suggests the instability of the reduced-CO complex in the absence of cholesterol.     

Laser intensity and wavelength dependence of Rose-Bengal-photosensitized inhibition of red blood cell acetylcholinesterase

The intensity and wavelength-dependence of Rose-Bengal-mediated photoinhibition of red blood cell acetylcholinesterase has been studied. Irradiation of dye-membrane suspensions with 308 nm laser excitation resulted in enzyme inhibition almost 50% greater than that obtained with 514 nm laser excitation. Sodium azide and argon purging greatly decreased the photosensitized enzyme inhibition at both wavelengths. Although Rose Bengal photosensitized enzyme inhibition more efficiently upon excitation into Sn (308 nm) than into S1 (514 nm), Stern-Volmer analysis of sodium azide quenching data gave similar quenching efficiencies at both wavelengths. Irradiation of dye-membrane suspensions with increasing intensities (Nd:YAG, 532 nm, 40 ps pulse duration) resulted in a decrease in enzyme inhibition. Saturation of the Rose Bengal fluorescence intensity and light transmission occurred with nearly the same intensity-dependence, suggesting that ground-state depletion occurs at the higher intensities. Our results demonstrate that excitation of a sensitizer into higher-lying excited singlet states can result in enhanced sensitizing efficiency. However, attempts to populate such states in Rose Bengal by sequential two-photon absorption using high intensities resulted only in ground-state depletion.     

Kinetics and mechanism of bilirubin binding to human serum albumin

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.     

Some differences between 470 nm and 510 nm in the acridine-orange-sensitized photodynamic actions on yeast cells

The two wavelengths that correspond to the absorption of two complexes formed in the interactions of acridine orange (AO) with nucleic acids were different in a few aspects of photodynamic action on yeast, Saccharomyces cerevisiae. (1) The dose-survival curve at 470 nm is not the same in shape as that at 510 nm. (2) The efficiency for the induction of gene conversion at 510 nm is higher than that obtained at 470 nm on the basis of both dose and survival.     

Investigation of the alpha(1)-glycine receptor channel-opening kinetics in the submillisecond time domain.

The activation and desensitization kinetics of the human alpha(1)-homooligomeric glycine receptor, which was transiently expressed in HEK 293 cells, were studied with a 100-microseconds time resolution to determine the rate and equilibrium constants of individual receptor reaction steps. Concentration jumps of the activating ligands glycine and beta-alanine were initiated by photolysis of caged, inactive precursors and were followed by neurotransmitter binding, receptor-channel opening, and receptor desensitization steps that were separated along the time axis. Analysis of the ligand concentration-dependence of these processes allows the determination of 1) the rate constants of glycine binding, k(+1) approximately 10(7) M(-1) s(-1), and dissociation, k(-1) = 1900 s(-1); 2) the rates of receptor-channel opening, k(op) = 2200 s(-1), and closing, k(cl) = 38 s(-1); 3) the receptor desensitization rate, alpha = 0.45 s(-1); 4) the number of occupied ligand binding sites necessary for receptor-channel activation and desensitization, n >/= 3; and 5) the maximum receptor-channel open probability, p(0) > 0.95. The kinetics of receptor-channel activation are insensitive to the transmembrane potential. A general model for glycine receptor activation explaining the experimental data consists of a sequential mechanism based on rapid ligand-binding steps preceding a rate-limiting receptor-channel opening reaction and slow receptor desensitization.     

Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein

Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma2P) increase rapidly at wavelengths below 800 nm, and the maximum sigma2P of LipDH is approximately 5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., approximately 410-490 nm and approximately 510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique.     

Spectral sensitivity studies on the visual system of the praying mantis, Tenodera sinensis

In these studies a constant ERG response was used as a measure of visual sensitivity to different wavelengths of light. The dark-adapted compound eye of Tenodera sinensis is dominated by a single class of photoreceptors. with a major peak of sensitivity at about 510–520 nm, and with a minor peak of sensitivity in the near-ultraviolet region at about 370 nm. The dark-adapted dorsal ocellus does not contain a homogeneous population of sensory receptors. The sensitivity function of the dark-adapted ocellus to longer wavelength light (yellow and red) is determined by a single receptor with a major peak of sensitivity in the green at 510–520 nm with some sensitivity in the near-ultraviolet. Sensitivity at shorter wavelengths (near-ultraviolet and blue), however, involves the stimulation of both this and a near-ultraviolet-sensitive receptor with a maximum sensitivity at about 370 nm. Anatomically, the sensory cells of the dorsal ocellus of Tenodera were determined histologically to be grouped into two distinct regions, each group making its own separate contribution to the ocellar nerve. This may represent the separation of two different photoreceptor types in the ocellus of the mantis.     

Quantum efficiencies of bacteriorhodopsin photochemical reactions.

Determination of quantum efficiencies of bacteriorhodopsin (bR) photoreactions is an essential step toward a full understanding of its light-driven proton-pumping mechanism. The bR molecules can be photoconverted into and from a K state, which is stable at 110 K. I measured the absorption spectra of pure bR, and the photoequilibrium states of bR and K generated with 420, 460, 500, 510, 520, 540, 560, 570, 580, 590, and 600 nm illumination at 110 K. The fraction of the K population in the photoequilibrium state, fk, is determined by AbR and AK the absorbances of the bR and K states at the excitation wavelengths, and also by phi 1 and phi 2, the quantum efficiencies for the bR to K and K to bR photoconversion: fK = phi 1 AbR/(phi 1AbR + phi 2Ak). By assuming that the ratio phi 1/phi 2 is the same at two different but close wavelengths, for example 570 and 580 nm, the value of phi 1/phi 2 at 570 and 580 nm was determined to be 0.55 +/- 0.02, and the spectrum of the K state was obtained with the peak absorbance at 607 nm. The values of phi 1/phi 2 at the other excitation wavelengths were then evaluated using the known K spectrum, and show almost no dependence on the excitation wavelength within the main band. The result phi 1/phi 2 = 0.55 +/- 0.02 disagrees with those of many other groups. The advantages of this method over others are its minimal assumptions and its straightforward procedure.     

Impairment of myocardial calcium homeostasis by antibodies against the adenine nucleotide translocator.

The adenine nucleotide translocator (ANT) is an autoantigen in myocarditis and dilated cardiomyopathy. Carrier-specific antibodies impair myocardial energy metabolism and heart function. They cross-react with a myolemmal calcium channel and alter calcium fluxes in isolated myocytes. To test whether antibodies against the ANT can alter calcium homeostasis in intact hearts, guinea pigs were immunized with the carrier protein and their isolated hearts loaded with the intracellular calcium indicator INDO-1. The diastolic and systolic ratios of fluorescence signals at 410 nm and 510 nm (emission wavelengths of the calcium-bound and calcium-free indicator), 'd-s410/510', were measured by excitation at 364 nm. This index of the transient calcium concentration associated with the contraction cycle correlated with the external heart work (EHW) in non-immunized controls. EHW of immunized animals was lower (76 +/- 62 vs 153 +/- 47 mJ/g/min in controls, p < 0.005) and the amplitude of d-s410/510 was elevated (27.6 +/- 4.1% of the average ratio of the whole heart cycle vs 21.7 +/- 1.2% in controls, p < 0.005) and essentially independent of EHW. Isoproterenol stimulation increased EHW in all hearts but d-s410/510 was hightened in control hearts, only. Thus, a disorder between cytosolic calcium transients and work was recorded in hearts from guinea pigs immunized with the ANT. It may contribute to an immunopathic mechanism of heart failure subsequent to myocarditis.