The 'gold standard' treatment of severe neonatal jaundice is phototherapy with blue-green light, which produces more polar photo-oxidation products that are easily excreted via the bile or urine. The aim of this study was to compare the effects of bilirubin (BR) and its major photo-oxidation product lumirubin (LR) on the proliferation, differentiation, morphology, and specific gene and protein expressions of self-renewing human pluripotent stem cell-derived neural stem cells (NSC). Neither BR nor LR in biologically relevant concentrations (12.5 and 25 µmol/L) affected cell proliferation or the cell cycle phases of NSC. Although none of these pigments affected terminal differentiation to neurons and astrocytes, when compared to LR, BR exerted a dose-dependent cytotoxicity on self-renewing NSC. In contrast, LR had a substantial effect on the morphology of the NSC, inducing them to form highly polar rosette-like structures associated with the redistribution of specific cellular proteins (β-catenin/N-cadherin) responsible for membrane polarity. This observation was accompanied by lower expressions of NSC-specific proteins (such as SOX1, NR2F2, or PAX6) together with the upregulation of phospho-ERK. Collectively, the data indicated that both BR and LR affect early human neurodevelopment in vitro, which may have clinical relevance in phototherapy-treated hyperbilirubinemic neonates.

• Publikační typ
• časopisecké články MeSH

For severe unconjugated hyperbilirubinemia the gold standard treatment is phototherapy with blue-green light, producing more polar photo-oxidation products, believed to be non-toxic. The aim of the present study was to compare the effects of bilirubin (BR) and lumirubin (LR), the major BR photo-oxidation product, on metabolic and oxidative stress markers. The biological activities of these pigments were investigated on several human and murine cell lines, with the focus on mitochondrial respiration, substrate metabolism, reactive oxygen species production, and the overall effects on cell viability. Compared to BR, LR was found to be much less toxic, while still maintaining a similar antioxidant capacity in the serum as well as suppressing activity leading to mitochondrial superoxide production. Nevertheless, due to its lower lipophilicity, LR was less efficient in preventing lipoperoxidation. The cytotoxicity of BR was affected by the cellular glycolytic reserve, most compromised in human hepatoblastoma HepG2 cells. The observed effects were correlated with changes in the production of tricarboxylic acid cycle metabolites. Both BR and LR modulated expression of PPARα downstream effectors involved in lipid and glucose metabolism. Proinflammatory effects of BR, evidenced by increased expression of TNFα upon exposure to bacterial lipopolysaccharide, were observed in murine macrophage-like RAW 264.7 cells. Collectively, these data point to the biological effects of BR and its photo-oxidation products, which might have clinical relevance in phototherapy-treated hyperbilirubinemic neonates and adult patients.

• Publikační typ
• časopisecké články MeSH

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

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

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

Although phototherapy was introduced as early as 1950's, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.

• MeSH
• bilirubin chemie   izolace a purifikace   farmakologie   MeSH
• buněčné linie MeSH
• buněčný cyklus účinky léků   MeSH
• cirkulární dichroismus MeSH
• fototerapie MeSH
• hem metabolismus   MeSH
• isomerie MeSH
• kinetika MeSH
• lidé MeSH
• ligandy MeSH
• regulace genové exprese účinky léků   MeSH
• sérový albumin metabolismus   MeSH
• spektrofotometrie ultrafialová MeSH
• světlo * MeSH
• viabilita buněk účinky léků   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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).

• 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
• 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