Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile

Biliverdin IX was purified from the bile of Atlantic salmon (Salmo salar) using a silica gel (Wakogel C-200) column. The yield was 49.5 mg per 100 ml of fresh bile and purity 95.3%. The biliverdin IX in the bile was quite stable when the bile was frozen at –80°C for a period of 40 days. However, 7.1% of the biliverdin IX was lost when the bile was stored at 4°C for 20 days. The purified biliverdin IX appeared as a single spot with R_f value of 0.25-0.27 on thin layer chromatography (TLC) and one main peak on high performance liquid chromatography (HPLC) at 436 or 650 nm. When the biliverdin IX was subjected to enzymic reduction with highly purified biliverdin reductase, two clear isobestic points were seen, at 384 and 670 nm. When the products of the reaction with biliverdin IX were extracted in butanol after completion of the reaction, one absorbance peak was observed at 468 nm. The time course of the reduction of biliverdin IX to bilirubin IX catalyzed by biliverdin reductase depended on reduced pyridine nucleotide. The time course of the NADPH-dependent reaction is different from that of the reaction with NADH. In the reduction of biliverdin IX , per mole of biliverdin IX reduced or per mole of bilirubin IX formed 1 mole of reduced pyridine nucleotide was consumed in both the NADH and NADPH systems.     

Heme oxygenase-1 induction may explain the antioxidant profile of pentaerythrityl trinitrate

The organic nitrate pentaerythrityl tetranitrate (PETN) is known to exert long-term antioxidant and antiatherogenic effects by as yet unidentified mechanisms. In cultured endothelial cells derived from human umbilical vein, the active PETN metabolite PETriN (0.01-1 mM) increased heme oxygenase (HO)-1 mRNA and protein levels in a concentration-dependent fashion. HO-1 induction was accompanied by a marked increase in catalytic activity of the enzyme as reflected by enhanced formation of carbon monoxide and bilirubin. Pretreatment with PETriN or bilirubin at low micromolar concentrations protected endothelial cells from hydrogen peroxide-mediated toxicity. HO-1 induction and endothelial protection by PETriN were not mimicked by isosorbide dinitrate, another long-acting nitrate. The present study demonstrates that PETriN stimulates mRNA and protein expression as well as enzymatic activity of the antioxidant defense protein HO-1 in endothelial cells. Increased HO-1 expression and ensuing formation of cytoprotective bilirubin may contribute to and explain the specific antioxidant and antiatherogenic actions of PETN.     

Electrically Contacted Bienzyme‐Functionalized Mesoporous Carbon Nanoparticle Electrodes: Applications for the Development of Dual Amperometric Biosensors and Multifuel‐Driven Biofuel Cells

The capping of electron relay units in mesoporous carbon nanoparticles (MPC NPs) by crosslinking of different enzymes on MPC NPs matrices leads to integrated electrically contacted bienzyme electrodes acting as dual biosensors or as functional bienzyme anodes and cathodes for biofuel cells. The capping of ferrocene methanol and methylene blue in MPC NPs by the crosslinking of glucose oxidase (GOx) and horseradish peroxidase (HRP) yields a functional sensing electrode for both glucose and H₂O₂, which also acts as a bienzyme cascaded system for the indirect detection of glucose. A MPC NP matrix, loaded with ferrocene methanol and capped by GOx/lactate oxidase (LOx), is implemented for the oxidation and detection of both glucose and lactate. Similarly, MPC NPs, loaded with 2,2′‐azino‐bis(3‐ethylbenzo­thiazoline‐6‐sulphonic acid), are capped with bilirubin oxidase (BOD) and catalase (Cat), to yield a bienzyme O₂ reduction cathode. A biofuel cell that uses the bienzyme GOx/LOx anode and the BOD/Cat cathode, glucose and/or lactate as fuels, and O₂ and/or H₂O₂ as oxidizers is assembled, revealing a power efficiency of ≈90 μW cm^?2 in the presence of the two fuels. The study demonstrates that multienzyme MPC NP electrodes may improve the performance of biofuel cells by oxidizing mixtures of fuels in biomass.     

Mutual structural effect of bilirubin and model membranes by vibrational circular dichroism

In this study, vibrational circular dichroism (VCD) spectroscopy was employed for the first time to study the bilirubin (BR) interaction with model membranes and models for membrane proteins. An enantioselective interaction of BR with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SPM) liposomes was observed by VCD and electronic circular dichroism (ECD) complemented by absorption and fluorescence spectroscopy. The M-form of BR was preferentially recognized in the BR/DMPC system at concentration above 1 × 10^− 4 M, for lower concentrations the P-form of BR was recognized by the DMPC liposomes. The VCD spectra also showed that the SPM liposomes, which represent the main component of nerve cell membrane, were significantly more disturbed by the presence of BR than the DMPC liposomes—a stable association with a strong VCD signal was observed providing the explanations for the supposed BR neurotoxicity. The effect of time and pH on the BR/DMPC or SPM liposome systems was shown to be essential while the effect of temperature in the range of 15–70 °C was negligible demonstrating the surprisingly high temperature stability of BR when interacting with the studied membranes. The influence of a membrane protein was tested on a model consisting of poly-l-arginine (PLAG) bound in the α-helical form to the surface of 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) liposomes and sodium dodecyl sulfate micelles. VCD and also ECD spectra showed that a variety of BR diastereoisomers interacted with PLAG in such systems. In a system of PLAG with micelles composed of sodium dodecyl sulfate, the M-form of bound BR was observed.     

Spectrofluorimetric determination of bilirubin in serum samples

A new spectrofluorimetric method was developed for determination of trace amount of bilirubin. Using oxytetracycline–Eu³⁺ as a fluorescent probe, in the buffer solution of pH = 7.3, bilirubin can reduce remarkably the fluorescence intensity of the oxytetracycline–Eu³⁺ complex at λ = 612 nm and the reduced fluorescence intensity was in proportion to the concentration of bilirubin. Optimum conditions for the determination of bilirubin were also investigated. The linear range and limit of detection for the determination of bilirubin were 5.0 × 10^?7, 3.0 × 10^?5 and 7.7 × 10^?8 mol L^?1, respectively. This method is simple, practical and relatively free of interference from coexisting substances and can be successfully applied to assess bilirubin in serum samples and compared with the modified Jendrassik–Grof method in clinical analysis. The quenching mechanism of the fluorescence intensity in the system is also discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of a novel duplication CFTRdup2 and functional impact of large rearrangements identified in the CFTR gene

The aim of this study was to investigate associations of two candidate gene SNPs of the endocannabinoid receptor type 1 gene (CNR1) with overweight, obesity and obesity-related traits in Chinese retired women. The study subjects were a subsample of the Taizhou Retiree Women Cohort, consisting of 2812 retired women aged 50-64 years recruited from Taizhou, Jiangsu, China. Neither rs2023239 nor rs806381 polymorphism was significantly associated with body mass index-defined overweight and obesity or waist-to-hip-ratio-defined obesity. For obesity-related traits, rs2023239 was significantly associated with glutamate pyruvate transaminase (GPT) (median, 18.00 vs 17.00 for TT and TC genotypes, respectively, P=0.043). The rs806381 also showed significant association with triglyceride (TG) (mean±SD, 1.46±0.20 vs 1.53±0.20 for GA and GG+AA genotypes, respectively, P=0.013) under the dominant genetic model. In conclusion, the rs2023239 and rs806381 polymorphisms of CNR1 were not associated with increased overweight and obesity risk. But the rs2023239 polymorphism was significantly associated with GPT, and the rs806381 polymorphism was significantly associated with TG.     

Effect of Bilirubin on Lipid Peroxidation, Sphingomyelinase Activity, and Apoptosis Induced by Sphingosine and UV Irradiation

The effect of bilirubin (BR) on sphingomyelin cycle activity, lipid peroxidation (LPO), and apoptosis induced by sphingosine and UV irradiation has been studied in vivo. Neutral Mg²⁺-dependent sphingomyelinase (SMase) activity and LPO level were monitored in heart, kidney, and liver of mice after administration of BR. BR inhibited both LPO and SMase activities in heart and kidney. BR induced a mild increase in LPO level and moderate increase in lipid contents in liver, consistent with the functional role of liver in both BR and lipid metabolism. BR injected to mice causes simultaneous and unidirectional alterations in both LPO level and SMase activity with a significant (p < 0.05) positive linear correlation between these two parameters. Sphingosine administration results in increased lipid peroxidation in murine liver. Data on DNA fragmentation indicate that exogenous BR may effectively protect thymus cells against sphingosine- and UV-mediated apoptosis. These results have revealed a biochemical association between oxidative stress and BR on one hand and the sphingomyelin cycle and apoptotic cell death on the other hand. Our data show that BR as an antioxidant, due to its effect on the sphingomyelin cycle, can protect membrane lipids against peroxidation and cells against apoptosis induced by various factors.     

Early predictive value of cord blood bilirubin and dynamic monitoring of transcutaneous bilirubin for hyperbilirubinemia of newborns

ObjectiveTo study the early predictive value of cord blood bilirubin and dynamic monitoring of transcutaneous bilirubin for hyperbilirubinemia of newborns.Methods389 newborns delivered from June 2014 to December 2015 were enrolled as the research subjects; detailed records were made about the general data of newborns and mothers, and after cord blood bilirubin being graded, the incidence of hyperbilirubinemia was counted, and the prediction efficiency of cord blood bilirubin was analyzed by receiver operator characteristic (ROC) curve. At the same time, the transcutaneous bilirubin was detected continuously when the neonate was born and 24 h, 48 h and 72 h after birth, and the relativity between transcutaneous bilirubin at 72 h and serum bilirubin was analyzed.ResultsNo significant difference was found in the hyperbilirubinemia group and the non-hyperbilirubinemia group concerning general data of the newborns and their mothers. With the concentration of cord blood bilirubin increased, the incidence of hyperbilirubinemia also increased; separate prediction of hyperbilirubinemia by cord blood bilirubin showed a sensitivity and specificity of 71.4% and 65.6% respectively, and they need further dynamic monitoring. The daily mean of transcutaneous bilirubin in hyperbilirubinemia group was significantly higher than that in non-hyperbilirubinemia group at 24 h, 48 h and 72 h, and the measurement value of transcutaneous bilirubin at 72 h had a high correlation with serum bilirubin. When transcutaneous bilirubin value is higher than 18, the incidence of hyperbilirubinemia should be considered.ConclusionsThe increase of cord blood bilirubin effectively predict the occurrence of neonatal hyperbilirubinemia. There is a good correlation between levels of transcutaneous bilirubin and serum bilirubin. Moreover, combined detection of transcutaneous bilirubin and cord blood bilirubin can significantly improve the prediction accuracy of hyperbilirubinemia.