Neonatal hyperbilirubinemia or jaundice is associated with kernicterus, resulting in permanent neurological damage or even death. Conventional phototherapy does not prevent hyperbilirubinemia or eliminate the need for exchange transfusion. Here we investigated the potential of therapeutic bile acids ursodeoxycholic acid (UDCA) and obeticholic acid (OCA, 6-α-ethyl-CDCA), a farnesoid-X-receptor (FXR) agonist, as preventive treatment options for neonatal hyperbilirubinemia using the hUGT1*1 humanized mice and Ugt1a-deficient Gunn rats. Treatment of hUGT1*1 mice with UDCA or OCA at postnatal days 10-14 effectively decreased bilirubin in plasma (by 82% and 62%) and brain (by 72% and 69%), respectively. Mechanistically, our findings indicate that these effects are mediated through induction of protein levels of hUGT1A1 in the intestine, but not in liver. We further demonstrate that in Ugt1a-deficient Gunn rats, UDCA but not OCA significantly decreases plasma bilirubin, indicating that at least some of the hypobilirubinemic effects of UDCA are independent of UGT1A1. Finally, using the synthetic, non-bile acid, FXR-agonist GW4064, we show that some of these effects are mediated through direct or indirect activation of FXR. Together, our study shows that therapeutic bile acids UDCA and OCA effectively reduce both plasma and brain bilirubin, highlighting their potential in the treatment of neonatal hyperbilirubinemia.

• MeSH
• bilirubin krev   MeSH
• ileum účinky léků   metabolismus   MeSH
• isoxazoly farmakologie   MeSH
• játra účinky léků   metabolismus   MeSH
• krysa rodu Rattus MeSH
• kyselina chenodeoxycholová analogy a deriváty   terapeutické užití   MeSH
• kyselina ursodeoxycholová terapeutické užití   MeSH
• myši MeSH
• novorozenecká hyperbilirubinemie krev   farmakoterapie   MeSH
• potkani Gunn MeSH
• receptory cytoplazmatické a nukleární agonisté   metabolismus   MeSH
• výsledek terapie MeSH
• žlučové kyseliny a soli terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bilirubin MeSH
• farnesoid X-activated receptor MeSH Prohlížeč
• GW 4064 MeSH Prohlížeč
• isoxazoly MeSH
• kyselina chenodeoxycholová MeSH
• kyselina ursodeoxycholová MeSH
• obeticholic acid MeSH Prohlížeč
• receptory cytoplazmatické a nukleární MeSH
• žlučové kyseliny a soli MeSH

BACKGROUND: Severe hyperbilirubinemia can cause permanent neurological damage in particular in neonates, whereas mildly elevated serum bilirubin protects from various oxidative stress-mediated diseases. The present work aimed to establish the intracellular unconjugated bilirubin concentrations (iUCB) thresholds differentiating between anti- and pro-oxidant effects. METHODS: Hepatic (HepG2), heart endothelial (H5V), kidney tubular (HK2) and neuronal (SH-SY5Y) cell lines were exposed to increasing concentration of bilirubin. iUCB, cytotoxicity, intracellular reactive oxygen species (ROS) concentrations, and antioxidant capacity (50% efficacy concentration (EC50)) were determined. RESULTS: Exposure of SH-SY5Y to UCB concentration > 3.6 µM (iUCB of 25 ng/mg) and >15 µM in H5V and HK2 cells (iUCB of 40 ng/mg) increased intracellular ROS production (p < 0.05). EC50 of the antioxidant activity was 21 µM (iUCB between 5.4 and 21 ng/mg) in HepG2 cells, 0.68 µM (iUCB between 3.3 and 7.5 ng/mg) in SH-SY5Y cells, 2.4 µM (iUCB between 3 and 6.7 ng/mg) in HK2 cells, and 4 µM (iUCB between 4.7 and 7.5 ng/mg) in H5V cells. CONCLUSIONS: In all the cell lines studied, iUCB of around 7 ng/mg protein had antioxidant activities, while iUCB > 25 ng/mg protein resulted in a prooxidant and cytotoxic effects. UCB metabolism was found to be cell-specific resulting in different iUCB.

• Klíčová slova
• ROS, antioxidant, bilirubin, bilirubin neurotoxicity, redox state,
• MeSH
• antioxidancia farmakologie   MeSH
• bilirubin farmakologie   MeSH
• hepatocelulární karcinom farmakoterapie   metabolismus   patologie   MeSH
• hyperbilirubinemie patofyziologie   MeSH
• lidé MeSH
• nádory jater farmakoterapie   metabolismus   patologie   MeSH
• neuroblastom farmakoterapie   metabolismus   patologie   MeSH
• oxidace-redukce MeSH
• oxidační stres účinky léků   MeSH
• oxidancia farmakologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antioxidancia MeSH
• bilirubin MeSH
• oxidancia MeSH
• reaktivní formy kyslíku 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
• Názvy látek
• bilirubin MeSH
• lumirubin MeSH Prohlížeč

Ectopic lipid accumulation in skeletal muscle and liver drives the pathogenesis of diabetes mellitus type 2 (DMT2). Mild hyperbilirubinaemia has been repeatedly suggested to play a role in the prevention of DMT2 and is known for its capacity to shape an improved lipid phenotype in humans and in animals. To date, the effect of bilirubin on lipid accumulation in tissues that are prone to ectopic lipid deposition is unclear. Therefore, we analyzed the effect of bilirubin on lipid accumulation in skeletal muscle and liver cell lines. C2C12 skeletal mouse muscle and HepG2 human liver cells were treated with physiological concentrations of free fatty acids (FFA) (0.5 mM and 1 mM) and unconjugated bilirubin (UCB) (17.1 and 55 µM). The intracellular presence of UCB upon exogenous UCB administration was confirmed by HPLC and the lipid accumulation was assessed by using Nile red. Exposure of both cell lines to UCB significantly reduced lipid accumulation by up to 23% (p ≤ 0.001) in HepG2 and by up to 17% (p ≤ 0.01) in C2C12 cells at 0.5 and 5 h under hypoglycaemic conditions. Simultaneously, UCB slightly increased FFA uptake in HepG2 cells after 0.5 and 5 h and in C2C12 cells after 12 h as confirmed by gas chromatographic analyses of the remaining FFA content in the incubation media. The effects of UCB on lipid accumulation and uptake were abolished in the presence of higher glucose concentrations. Monitoring the uptake of a radiolabeled glucose analogue [18F]FDG: (2-deoxy-2-[18F]fluoro-D-glucose) into both cell types further indicated higher glucose consumption in the presence of UCB. In conclusion, our findings show that UCB considerably decreases lipid accumulation in skeletal muscle and liver cells within a short incubation time of max. 5 h which suggests that mildly elevated bilirubin levels could lower ectopic lipid deposition, a major key element in the pathogenesis of DMT2.

• Klíčová slova
• C2C12 skeletal muscle cells, HepG2 cells, [18F]FDG uptake, bilirubin, ectopic lipid accumulation, insulin resistance, lipid accumulation, mild hyperbilirubinaemia,
• Publikační typ
• časopisecké články MeSH

Decreased inflammatory status has been reported in subjects with mild unconjugated hyperbilirubinemia. However, mechanisms of the anti-inflammatory actions of bilirubin (BR) are not fully understood. The aim of this study is to assess the role of BR in systemic inflammation using hyperbilirubinemic Gunn rats as well as their normobilirubinemic littermates and further in primary hepatocytes. The rats were treated with lipopolysaccharide (LPS, 6 mg/kg intraperitoneally) for 12 h, their blood and liver were collected for analyses of inflammatory and hepatic injury markers. Primary hepatocytes were treated with BR and TNF-α. LPS-treated Gunn rats had a significantly decreased inflammatory response, as evidenced by the anti-inflammatory profile of white blood cell subsets, and lower hepatic and systemic expressions of IL-6, TNF-α, IL-1β, and IL-10. Hepatic mRNA expression of LPS-binding protein was upregulated in Gunn rats before and after LPS treatment. In addition, liver injury markers were lower in Gunn rats as compared to in LPS-treated controls. The exposure of primary hepatocytes to TNF-α with BR led to a milder decrease in phosphorylation of the NF-κB p65 subunit compared to in cells without BR. In conclusion, hyperbilirubinemia in Gunn rats is associated with an attenuated systemic inflammatory response and decreased liver damage upon exposure to LPS.

• Klíčová slova
• Gunn rats, LPS, NF-κB, bilirubin, hyperbilirubinemia, inflammation,
• MeSH
• apoptóza účinky léků   MeSH
• bilirubin farmakologie   MeSH
• biologické markery krev   MeSH
• cytokiny krev   genetika   metabolismus   MeSH
• cytoprotekce účinky léků   MeSH
• fosforylace účinky léků   MeSH
• hepatocyty metabolismus   MeSH
• hyperbilirubinemie krev   komplikace   MeSH
• játra metabolismus   MeSH
• kultivované buňky MeSH
• leukocyty metabolismus   MeSH
• lipopolysacharidy MeSH
• messenger RNA genetika   metabolismus   MeSH
• NF-kappa B metabolismus   MeSH
• potkani Gunn MeSH
• signální transdukce MeSH
• zánět komplikace   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bilirubin MeSH
• biologické markery MeSH
• cytokiny MeSH
• lipopolysacharidy MeSH
• messenger RNA MeSH
• NF-kappa B MeSH

Bilirubin is considered to be one of the most potent endogenous antioxidants in humans. Its serum concentrations are predominantly affected by the activity of hepatic bilirubin UDP-glucuronosyl transferase (UGT1A1). Our objective was to analyze the potential bilirubin-modulating effects of natural polyphenols from milk thistle (Silybum marianum), a hepatoprotective herb. Human hepatoblastoma HepG2 cells were exposed to major polyphenolic compounds isolated from milk thistle. Based on in vitro studies, 2,3-dehydrosilybins A and B were selected as the most efficient compounds and applied either intraperitoneally or orally for seven days to C57BL/6 mice. After, UGT1A1 mRNA expression, serum, intrahepatic bilirubin concentrations, and lipoperoxidation in the liver tissue were analyzed. All natural polyphenols used increased intracellular concentration of bilirubin in HepG2 cells to a similar extent as atazanavir, a known bilirubinemia-enhancing agent. Intraperitoneal application of 2,3-dehydrosilybins A and B (the most efficient flavonoids from in vitro studies) to mice (50 mg/kg) led to a significant downregulation of UGT1A1 mRNA expression (46 ± 3% of controls, p < 0.005) in the liver and also to a significant increase of the intracellular bilirubin concentration (0.98 ± 0.03vs.1.21 ± 0.02 nmol/mg, p < 0.05). Simultaneously, a significant decrease of lipoperoxidation (61 ± 2% of controls, p < 0.005) was detected in the liver tissue of treated animals, and similar results were also observed after oral treatment. Importantly, both application routes also led to a significant elevation of serum bilirubin concentrations (125 ± 3% and 160 ± 22% of the controls after intraperitoneal and oral administration, respectively, p < 0.005 in both cases). In conclusion, polyphenolic compounds contained in silymarin, in particular 2,3-dehydrosilybins A and B, affect hepatic and serum bilirubin concentrations, as well as lipoperoxidation in the liver. This phenomenon might contribute to the hepatoprotective effects of silymarin.

• MeSH
• bilirubin metabolismus   MeSH
• buňky Hep G2 MeSH
• flavonoidy chemie   izolace a purifikace   farmakologie   MeSH
• glukuronosyltransferasa genetika   metabolismus   MeSH
• hemoxygenasa-1 genetika   metabolismus   MeSH
• intracelulární prostor metabolismus   MeSH
• játra účinky léků   metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• peroxidace lipidů účinky léků   MeSH
• regulace genové exprese enzymů účinky léků   MeSH
• silibinin aplikace a dávkování   farmakologie   MeSH
• silymarin izolace a purifikace   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bilirubin MeSH
• flavonoidy MeSH
• glukuronosyltransferasa MeSH
• hemoxygenasa-1 MeSH
• HMOX1 protein, human MeSH Prohlížeč
• messenger RNA MeSH
• silibinin MeSH
• silymarin MeSH
• UGT1A1 enzyme MeSH Prohlížeč

Mild constitutive hyperbilirubinemia is associated with a reduced risk of cardiovascular diseases, diabetes, and cancer. Since these pathologies are associated with aging, inflammation, and oxidative stress, we investigated whether hyperbilirubinemia interferes with ROS homeostasis in cell cultures and with inflammation, senescence, and mitochondrial dysfunction in aged rats. Human embryonic kidney cells and rat primary fibroblasts showed a dose-dependent decrease in the ratio of oxidized/reduced glutathione, intracellular H2O2 levels, and mitochondrial ROS production, with increasing bilirubin concentrations in the culture media. Compared to their normobilirubinemic siblings, aged hyperbilirubinemic Gunn rats showed significantly smaller amounts of visceral fat, better glucose tolerance, and decreased serum levels of proinflammatory cytokines TNFα, IL-1β, and IL-18. Simultaneously, livers from Gunn rats showed decreased expression of senescence markers and cell cycle inhibitors p21 and p16. Mitochondria from aged Gunn rats showed higher respiration and lower H2O2 production compared to controls. In conclusion, we demonstrated that mildly elevated serum bilirubin is generally associated with attenuation of oxidative stress and with better anthropometric parameters, decreased inflammatory status, increased glucose tolerance, fewer signs of cellular senescence, and enhanced mitochondrial function in aged rats.

• MeSH
• bilirubin krev   MeSH
• fibroblasty metabolismus   patologie   MeSH
• hyperbilirubinemie krev   patologie   MeSH
• intracelulární prostor metabolismus   MeSH
• kultivované buňky MeSH
• metabolické nemoci komplikace   patologie   MeSH
• mitochondrie metabolismus   MeSH
• potkani Gunn MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• stárnutí patologie   MeSH
• zánět komplikace   patologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bilirubin MeSH
• reaktivní formy kyslíku MeSH

Therapies to prevent severe neonatal unconjugated hyperbilirubinemia and kernicterus are phototherapy and, in unresponsive cases, exchange transfusion, which has significant morbidity and mortality risks. Neurotoxicity is caused by the fraction of unconjugated bilirubin not bound to albumin (free bilirubin, Bf). Human serum albumin (HSA) administration was suggested to increase plasma bilirubin-binding capacity. However, its clinical use is infrequent due to difficulties to address its potential preventive and curative benefits, and to the absence of reliable markers to monitor bilirubin neurotoxicity risk. We used a genetic mouse model of unconjugated hyperbilirubinemia showing severe neurological impairment and neonatal lethality. We treated mutant pups with repeated HSA administration since birth, without phototherapy application. Daily intraperitoneal HSA administration completely rescued neurological damage and lethality, depending on dosage and administration frequency. Albumin infusion increased plasma bilirubin-binding capacity, mobilizing bilirubin from tissues to plasma. This resulted in reduced plasma Bf, forebrain and cerebellum bilirubin levels. We showed that, in our experimental model, Bf is the best marker to determine the risk of developing neurological damage. These results support the potential use of albumin administration in severe acute hyperbilirubinemia conditions to prevent or treat bilirubin neurotoxicity in situations in which exchange transfusion may be required.

• MeSH
• bilirubin krev   MeSH
• fototerapie metody   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• mozeček účinky léků   MeSH
• myši MeSH
• nemoci nervového systému etiologie   prevence a kontrola   MeSH
• novorozenecká hyperbilirubinemie krev   komplikace   MeSH
• novorozenecká žloutenka krev   komplikace   MeSH
• sérový albumin aplikace a dávkování   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bilirubin MeSH
• sérový albumin MeSH

High plasma concentrations of bile acids (BA) and bilirubin are hallmarks of cholestasis. BA are implicated in the pathogenesis of cholestatic liver damage through mechanisms involving oxidative stress, whereas bilirubin is a strong antioxidant. We evaluated the roles of bilirubin and BA on mediating oxidative stress in rats following bile duct ligation (BDL). Adult female Wistar and Gunn rats intraperitoneally anaesthetized with ketamine and xylazine underwent BDL or sham operation. Cholestatic markers, antioxidant capacity, lipid peroxidation and heme oxygenase (HO) activity were determined in plasma and/or liver tissue 5 days after surgery. HepG2-rNtcp cells were used for in vitro experiments. Plasma bilirubin levels in control and BDL animals positively correlated with plasma antioxidant capacity. Peroxyl radical scavenging capacity was significantly higher in the plasma of BDL Wistar rats (210 ± 12%, P < 0.0001) compared to controls, but not in the liver tissues. Furthermore after BDL, lipid peroxidation in the livers increased (179 ± 37%, P < 0.01), whereas liver HO activity significantly decreased to 61% of control levels (P < 0.001). Addition of taurocholic acid (TCA, ≥ 50 μmol/l) to liver homogenates increased lipid peroxidation (P < 0.01) in Wistar, but not in Gunn rats or after the addition of bilirubin. In HepG2-rNtcp cells, TCA decreased both HO activity and intracellular bilirubin levels. We conclude that even though plasma bilirubin is a marker of cholestasis and hepatocyte dysfunction, it is also an endogenous antioxidant, which may counteract the pro-oxidative effects of BA in circulation. However, in an animal model of obstructive cholestasis, we found that BA compromise intracellular bilirubin levels making hepatocytes more susceptible to oxidative damage.

• MeSH
• bilirubin metabolismus   MeSH
• cholestáza metabolismus   patologie   MeSH
• hemová oxygenasa (decyklizující) krev   MeSH
• intracelulární prostor metabolismus   MeSH
• játra metabolismus   patologie   MeSH
• krysa rodu Rattus MeSH
• kyselina taurocholová farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• oxidační stres účinky léků   MeSH
• peroxidace lipidů účinky léků   MeSH
• potkani Gunn MeSH
• potkani Wistar MeSH
• žlučové kyseliny a soli metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bilirubin MeSH
• hemová oxygenasa (decyklizující) MeSH
• kyselina taurocholová MeSH
• žlučové kyseliny a soli MeSH