Although phototherapy (PT) is a standard treatment for neonatal jaundice, no validated clinical methods for determination of bilirubin phototherapy products are available. Thus, the aim of our study was to establish a such method for clinical use. To achieve this aim, a LC-MS/MS assay for simultaneous determination of Z-lumirubin (LR) and unconjugated bilirubin (UCB) was conducted. LR was purified after irradiation of UCB at 460 nm. The assay was tested on human sera from PT-treated neonates. Samples were separated on a HPLC system with a triple quadrupole mass spectrometer detector. The instrument response was linear up to 5.8 and 23.4 mg/dL for LR and UCB, respectively, with submicromolar limits of detection and validity parameters relevant for use in clinical medicine. Exposure of newborns to PT raised serum LR concentrations three-fold (p < 0.01), but the absolute concentrations were low (0.37 ± 0.16 mg/dL), despite a dramatic decrease of serum UCB concentrations (13.6 ± 2.2 vs. 10.3 ± 3.3 mg/dL, p < 0.01). A LC-MS/MS method for the simultaneous determination of LR and UCB in human serum was established and validated for clinical use. This method should help to monitor neonates on PT, as well as to improve our understanding of both the kinetics and biology of bilirubin phototherapy products.
- MeSH
- bilirubin analogy a deriváty krev chemie MeSH
- chromatografie kapalinová MeSH
- fototerapie metody MeSH
- lidé MeSH
- molekulární struktura MeSH
- novorozenec MeSH
- novorozenecká žloutenka krev terapie MeSH
- sérum chemie MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bilirubin MeSH
- lumirubin MeSH Prohlížeč
BACKGROUND: The action spectrum for bilirubin photodegradation has been intensively studied. However, questions still remain regarding which light wavelength most efficiently photodegrades bilirubin. In this study, we determined the in vitro effects of different irradiation wavelength ranges on bilirubin photodegradation. METHODS: In our in vitro method, normalized absolute irradiance levels of 4.2 × 1015 photons/cm2/s from light-emitting diodes (ranging from 390-530 nm) and 10-nm band-pass filters were used to irradiate bilirubin solutions (25 mg/dL in 4% human serum albumin). Bilirubin and its major photoisomer concentrations were determined; the half-life time of bilirubin (t1/2) was calculated for each wavelength range, and the spectral characteristics for bilirubin photodegradation products were obtained for key wavelengths. RESULTS: The in vitro photodegradation of bilirubin at 37 °C decreased linearly as the wavelength was increased from 390 to 500 nm with t1/2 decreasing from 63 to 17 min, respectively. At 460 ± 10 nm, a significantly lower rate of photodegradation and thus higher t1/2 (31 min) than that at 500 nm (17 min) was demonstrated. CONCLUSION: In our system, the optimum bilirubin photodegradation and lumirubin production rates occurred between 490 and 500 nm. Spectra shapes were remarkably similar, suggesting that lumirubin production was the major process of bilirubin photodegradation.
- MeSH
- bilirubin analogy a deriváty krev chemie účinky záření MeSH
- fotolýza účinky záření MeSH
- fototerapie metody MeSH
- isomerie MeSH
- lidé MeSH
- lidský sérový albumin chemie účinky záření MeSH
- novorozenec MeSH
- novorozenecká hyperbilirubinemie krev terapie MeSH
- spektrofotometrie MeSH
- světlo * MeSH
- techniky in vitro MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bilirubin MeSH
- lidský sérový albumin MeSH
- lumirubin MeSH Prohlížeč
Phototherapy was introduced in the early 1950's, and is the primary treatment of severe neonatal jaundice or Crigler-Najjar syndrome. Nevertheless, the potential biological effects of the products generated from the photodegradation of bilirubin during phototherapy remain unknown. This is very relevant in light of recent clinical observations demonstrating that the use of aggressive phototherapy can increase morbidity or even mortality, in extremely low birthweight (ELBW) infants. The aim of our study was to investigate the effects of bilirubin, lumirubin (LR, its major photo-oxidative product), and BOX A and B (its monopyrrolic oxidative products) on the central nervous system (CNS) using in vitro and ex vivo experimental models. The effects of bilirubin photoproducts on cell viability and expression of selected genes were tested in human fibroblasts, three human CNS cell lines (neuroblastoma SH-SY5Y, microglial HMC3, and glioblastoma U-87 cell lines), and organotypic rat hippocampal slices. Neither bilirubin nor its photo-oxidative products affected cell viability in any of our models. In contrast, LR in biologically-relevant concentrations (25 μM) significantly increased gene expression of several pro-inflammatory genes as well as production of TNF-α in organotypic rat hippocampal slices. These findings might underlie the adverse outcomes observed in ELBW infants undergoing aggressive phototherapy.
- MeSH
- bilirubin analogy a deriváty imunologie MeSH
- buněčné linie MeSH
- fotolýza MeSH
- fototerapie škodlivé účinky MeSH
- hipokampus imunologie patologie MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- novorozenec MeSH
- novorozenecká žloutenka terapie MeSH
- potkani Wistar MeSH
- TNF-alfa imunologie MeSH
- viabilita buněk MeSH
- zánět imunologie patologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- novorozenec MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bilirubin MeSH
- lumirubin MeSH Prohlížeč
- TNF-alfa MeSH
The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp-HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment-HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA).
- Klíčová slova
- Antioxidant, Bilirubin conversion, Bilirubin–biliverdin reversible antioxidant redox cycle, Circular dichroism, Molecular docking, Photo-isomerization,
- MeSH
- bilirubin analogy a deriváty chemie metabolismus MeSH
- biliverdin analogy a deriváty chemie metabolismus MeSH
- cirkulární dichroismus MeSH
- fluorescenční spektrometrie MeSH
- fotochemické procesy * MeSH
- kompetitivní vazba MeSH
- lidé MeSH
- lidský sérový albumin MeSH
- ligandy MeSH
- molekulární konformace MeSH
- molekulární modely * MeSH
- oxidace-redukce MeSH
- sérový albumin chemie metabolismus MeSH
- simulace molekulového dockingu MeSH
- stereoizomerie MeSH
- taurin analogy a deriváty chemie metabolismus MeSH
- tryptofan chemie MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ALB protein, human MeSH Prohlížeč
- bilirubin ditaurine MeSH Prohlížeč
- bilirubin MeSH
- biliverdin MeSH
- lidský sérový albumin MeSH
- ligandy MeSH
- lumirubin MeSH Prohlížeč
- sérový albumin MeSH
- taurin MeSH
- tryptofan MeSH
- xanthobilirubic acid MeSH Prohlížeč
Antioxidant, anti-inflammatory and anti-atherogenic effects have been associated with elevations of unconjugated bilirubin (UCB) in serum and with the induction of heme oxygenase-1 (HO-1), the rate-limiting enzyme in UCB synthesis. The aim of this study was to investigate the intracellular metabolism and antioxidant properties of UCB in human hepatoblastoma HepG2 cells and tissues of Wistar rats exposed to oxidative stressors and lipopolysaccharide (LPS), respectively. Intracellular UCB concentrations in HepG2 cells correlated with its levels in culture media (p < 0.001) and diminished lipid peroxidation in a dose-dependent manner (p < 0.001). Moreover, induction of HO-1 with sodium arsenite led to 2.4-fold (p = 0.01) accumulation of intracellular UCB over basal level while sodium azide-derived oxidative stress resulted in a 60% drop (p < 0.001). This decrease was ameliorated by UCB elevation in media or by simultaneous induction of HO-1. In addition, hyperbilirubinemia and liver HO-1 induction in LPS-treated rats resulted in a 2-fold accumulation of tissue UCB (p = 0.01) associated with enhanced protection against lipid peroxidation (p = 0.02). In conclusion, hyperbilirubinemia and HO-1 induction associated with inflammation and oxidative stress increase intracellular concentrations of UCB, thus enhancing the protection of cellular lipids against peroxidation. Therefore, the previously reported protective effects of hyperbilirubinemia and HO-1 induction are at least in part due to intracellular accumulation of UCB.
- MeSH
- aktivace enzymů účinky léků MeSH
- antioxidancia metabolismus MeSH
- arsenitany farmakologie MeSH
- bilirubin aplikace a dávkování analogy a deriváty metabolismus MeSH
- buňky Hep G2 MeSH
- hem analýza MeSH
- hemoxygenasa-1 metabolismus MeSH
- hyperbilirubinemie metabolismus MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- lipopolysacharidy aplikace a dávkování MeSH
- methemalbumin farmakologie MeSH
- oxidační stres účinky léků MeSH
- peroxidace lipidů účinky léků MeSH
- potkani Wistar MeSH
- sloučeniny sodíku farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antioxidancia MeSH
- arsenitany MeSH
- bilirubin MeSH
- hem MeSH
- hemoxygenasa-1 MeSH
- HMOX1 protein, human MeSH Prohlížeč
- lipopolysacharidy MeSH
- methemalbumin MeSH
- sloučeniny sodíku MeSH
- sodium arsenite MeSH Prohlížeč
Bilirubin is degraded in the human gut by microflora into urobilinoids. In our study we investigated whether the bilirubin-reducing strain of Clostridium perfringens can reduce bilirubin ditaurate (BDT), a bile pigment of some lower vertebrates, without hydrolysis of the taurine moiety. C. perfringes was incubated under anaerobic conditions with BDT; reduction products were quantified by spectrophotometry and separated by TLC. Based on Rf values of BDT reduction products and synthetic urobilinogen ditaurate, three novel taurine-conjugated urobilinoids were identified. It is likely that bilirubin-reducing enzyme(s) serve for the effective disposal of electrons produced by fermentolytic processes in these anaerobic bacteria.
- MeSH
- bilirubin analogy a deriváty izolace a purifikace metabolismus MeSH
- chromatografie na tenké vrstvě MeSH
- Clostridium perfringens izolace a purifikace metabolismus MeSH
- feces mikrobiologie MeSH
- hydrolýza MeSH
- lidé MeSH
- novorozenec MeSH
- oxidace-redukce MeSH
- střeva mikrobiologie MeSH
- taurin analogy a deriváty izolace a purifikace metabolismus MeSH
- urobilinogen MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bilirubin ditaurine MeSH Prohlížeč
- bilirubin MeSH
- taurin MeSH
- urobilinogen MeSH
Bilirubin ditaurate (taurobilirubin) proved to be very suitable for estimation of efficiency of different photosensitizers and for determination of the activity of singlet oxygen. The degree of its bleaching was measured at 450 nm, the excitation light was over 600 nm. Halogen lamps were used as light sources. The decoloration of bilirubin ditaurate was related to the intensity of the light source in the presence of the photosensitizer meso-tetra (4-sulfonatophenyl)-porphine (TPPS4). From photosensitizers conventionally used in photodynamic therapy (photosan 3, TPPS4, and Zn and Al chelates of phthalocyanine) chloroaluminium phthalocyanine was most effective. From synthetic dyes of nonporphyrin type thiazine dyes were most active. The presumption is expressed that the efficiency of noncoherent light is comparable to the laser radiation when photosensitizers with large absorption band 600-700 nm are used for photodynamic therapy.
- MeSH
- bilirubin analogy a deriváty chemie MeSH
- fotochemie MeSH
- fotochemoterapie MeSH
- fotosenzibilizující látky chemie MeSH
- taurin analogy a deriváty chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bilirubin MeSH
- fotosenzibilizující látky MeSH
- taurin MeSH
- taurobilirubin MeSH Prohlížeč
Oxygen concentration controls the reaction rate of porphyrin-sensitized photo-oxidation under constant experimental conditions. The rate of bilirubin and ditaurobilirubin auto-oxidation and meso-tetra-(4-sulphonatophenyl) porphine-sensitized photo-oxidation in aqueous solution increases by about 3.5 times in 0.5 MPa of oxygen. Use of hyperbaric oxygenation in the photodynamic therapy of tumours and in the phototherapy of jaundice is proposed.
- MeSH
- bilirubin * analogy a deriváty MeSH
- fotochemoterapie MeSH
- fototerapie MeSH
- hyperbarická oxygenace * MeSH
- kyslík * MeSH
- lidé MeSH
- oxidace-redukce MeSH
- porfyriny * MeSH
- radiosenzibilizující látky * MeSH
- taurin analogy a deriváty MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bilirubin * MeSH
- ditaurobilirubin MeSH Prohlížeč
- kyslík * MeSH
- porfyriny * MeSH
- radiosenzibilizující látky * MeSH
- taurin MeSH
- tetraphenylporphine sulfonate MeSH Prohlížeč
In 0.05--0.1 mmol.l-1 concentration, bilirubin inhibits ADP-activated respiration of isolated liver mitochondria; it has no effect on respiration in the absence of ADP. Bilirubin-induced inhibition of respiration is not abolished by serum albumin, but bilirubin bound to serum albumin and the photodegradation products of bilirubin have no inhibitory effect.
- MeSH
- adenosindifosfát farmakologie MeSH
- bilirubin analogy a deriváty farmakologie MeSH
- fotolýza MeSH
- jaterní mitochondrie metabolismus MeSH
- krysa rodu Rattus MeSH
- sérový albumin farmakologie MeSH
- spotřeba kyslíku účinky léků MeSH
- sukcináty metabolismus MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adenosindifosfát MeSH
- bilirubin MeSH
- sérový albumin MeSH
- sukcináty MeSH
- MeSH
- barvicí látky metabolismus MeSH
- bilirubin analogy a deriváty metabolismus MeSH
- glukuronosyltransferasa analýza MeSH
- jaterní testy metody MeSH
- játra metabolismus MeSH
- krysa rodu Rattus MeSH
- sulfobromoftalein metabolismus MeSH
- taurin analogy a deriváty metabolismus MeSH
- transportní proteiny metabolismus MeSH
- žluč metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- barvicí látky MeSH
- bilirubin MeSH
- glukuronosyltransferasa MeSH
- sulfobromoftalein MeSH
- taurin MeSH
- taurobilirubin MeSH Prohlížeč
- transportní proteiny MeSH
