Amperometric detection of bilirubin from a micro-sensing electrode with a synthetic bilirubin imprinted poly(MAA-co-EGDMA) film

Poly(methacrylic acid-co-ethyl glycol dimethylacrylate) (poly(MAA-co-EGDMA)) imprinted with alpha-bilirubin was shown to be able to bind alpha-bilirubin in our previous work. In this work, the corresponding imprinted polymer thin film was synthesized onto a thiol treated Au electrode by surface grafting polymerization. Bilirubin was able to be detected by an Au electrode, however, the electrode was not be able to discriminate bilirubin from the other matrix components if clinical samples were used. Therefore, the imprinted material was introduced so that the modified Au electrode could specifically detect bilirubin. Optimal potential was found to be 0.55 V and this was set for the rest of experiments. The imprinting factor of 3.16 was confirmed by comparing the signals from the MIP-Au and the NIP (non-imprinted polymer)-Au electrode. Calibration of the bilirubin concentration with respect to the current by the MIP-Au electrode was made within the range of 5mg/dl and a detection sensitivity of 0.644 microA/mg/dl (2.58 microA/cm(2)/mg/dl) was obtained. Furthermore, a linear correlation of the bilirubin concentration within 1.0mg/dl versus detection current was also achieved. Bilirubin was further detected by the MIP-Au electrode in the presence of fetal bovine serum (FBS). Repeated detection of bilirubin with at least three detection batches was performed and the reproducibility of the same piece of MIP-Au electrode was confirmed. The result was compared to those obtained from the serum and the solvent solution. The results indicated the feasibility of using the bilirubin imprinted poly(MAA-co-EGDMA) film as a sensing electrode for the clinical detection of bilirubin in serum.     

Comparison of hepatic, renal and intestinal bilirubin UDP-glucuronyl transferase activities in rat microsomes

1. Bilirubin UDP-glucuronyltransferase activity and its dependence on substrate concentrations in rat liver, renal cortex and intestinal mucosa microsomes were studied. 2. Bilirubin monoglucuronide synthesis from unconjugated bilirubin was a higher capacity, lower affinity step in comparison with bilirubin diglucuronide formation in the three tissues tested. 3. Bilirubin glucuronide formation in liver microsomes showed a higher capacity but a lower affinity than extrahepatic ones. Renal cortex and intestinal mucosa exhibited similar kinetics parameters. 4. In vitro bilirubin glucuronidation in renal cortex and intestinal mucosa was quantitatively important as compared with the hepatic one.     

New sorbent for bilirubin removal from human plasma: Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads

Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads were investigated as a specific sorbent for bilirubin removal from human plasma. The poly(EGDMA–HEMA) microbeads were prepared by a modified suspension copolymerization technique. Cibacron Blue F3GA was covalently coupled to the poly(EGDMA–HEMA) microbeads via the nucleophilic reaction between the chloride of its triazine ring and the hydroxyl groups of the HEMA molecule, under alkaline conditions. Bilirubin adsorption was investigated from hyperbilirubinemic human plasma on the poly(EGDMA–HEMA) microbeads containing different amounts of immobilized Cibacron Blue F3GA, (between 5.0–16.5 μmol/g). The non-specific bilirubin adsorption on the unmodified poly(EGDMA–HEMA) microbeads were 0.32 mg/g from human plasma. Higher bilirubin adsorption values, up to 14.8 mg/g, were obtained with the Cibacron Blue F3GA-immobilized microbeads. Bilirubin molecules interacted with these sorbents directly. Contribution of albumin adsorption on the bilirubin adsorption was pronounced. Bilirubin adsorption increased with increasing temperature.     

Does bilirubin play a role in the pathogenesis of both cholesterol and pigment gallstone formation? Direct and indirect influences of bilirubin on bile lithogenicity.

Bilirubin is found in the center of cholesterol gallstones, but its pathogenic role in their formation is unknown. Bilirubin causes a disproportionate reduction of biliary lipid secretion without affecting bile salt secretion in association with a change of biliary lecithin species, which modulates the cholesterol crystallization process. Therefore, the present study investigated whether bilirubin can influence the cholesterol crystallization procedure, and the mechanism(s) of any such action. Supersaturated model bile was prepared (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 9.0 g/dl, and cholesterol saturation index of 1.8), and cholesterol crystallization was monitored over time using a spectrophotometer and video-enhanced differential contrast microscopy in the absence or presence of bilirubin (at a final concentration of 10 microM, 20 microM, 40 microM, and 100 microM). Bilirubin enhanced the onset of cholesterol crystallization by 50%, whereas the crystal growth rate and final crystal mass were reduced at a high concentration of bilirubin. Taken together, these results suggest that bilirubin influences the cholesterol crystallization process, by either a direct interaction with biliary lipids that alters metastability, an indirect alteration of the bile salt-micellar lipid holding capacity, or both. Thus, bilirubin may play a role in the pathogenesis of both cholesterol and pigment gallstones.     

胆红素对人胎盘谷胱甘肽S-转移酶的别构效应

胆红素是人胎盘谷胱甘肽S—转移酶(GST—π)的别构效应剂,在胆红素存在下,底物谷胱甘肽(GSH)呈同促正协同效应:胆红素浓度愈高,Hill氏系数(n_H)也愈大,胆红素本身对酶的结合也呈同促正协同效应。胆红素还能加速GST-π在缺乏疏基保护剂时的自然失活,加速GST-π的氨基被2,4,6—三硝基苯磺酸(TNBS)、胍基被丁二酮以及羧基被N-乙基-N’-(3-二甲胺基丙基)羧二亚胺(EDC)的修饰,但却抑制N-乙基顺丁二酰亚胺(NEMI)对疏基的修饰,胆红素这种对失活作用的影响可能和胆红素引起GST-π空间构象的变化有关,对其他可能性也作了讨论。     

Bilirubin Inhibits Tumor Cell Growth via Activation of ERK

Bilirubin for decades was considered a potentially toxic waste product of heme degradation until the discovery that it is a potent antioxidant. Accumulating data from observations in humans and experimental studies indicate that the bile pigment may be protective against certain diseases. Based on our own observations that bilirubin induces cell cycle arrest in abnormally proliferating vascular smooth muscle cells and clinical observations describing a lesser incidence of cancer in healthy individuals with high normal or slightly elevated serum bilirubin levels, we hypothesized that bilirubin might suppress tumor cell proliferation in vitro and in vivo. As possible effectors we analyzed key proteins that are involved in cell cycle progression and apoptosis. In vivo tumor growth was assessed in BALB/c nude mice bearing HRT-18 colon cancer xenografts that were treated with bilirubin. In vitro, we investigated the effect of bilirubin on various cell lines and the signaling pathways involved in bilirubin action on tumor cell proliferation in HRT-18 cells using western blots. Bilirubin potently inhibited tumor cell proliferation in vivo and acted cytostatic and pro-apoptotic in vitro. The signaling cascades responsible for this action involved induction of p53, p27, hypophosphorylation of the retinoblastoma tumor suppressor protein as well as caspase activation. These effects were dependent on ERK 1/2. Our study demonstrates that bilirubin may play a role in the defense against cancer by interfering with pro-cancerogenic signaling pathways.     

Enhanced oxidation of bilirubin by an immobilized tri-enzyme system of glucose oxidase, bilirubin oxidase and horseradish peroxidase

Bilirubin oxidase was immobilized to nylon fibres. A tri-enzyme system composed of glucose oxidase, bilirubin oxidase and horseradish peroxidase was also immobilized to the fibres. Both immobilized systems were tested and it was found that the latter gave enhanced oxidation rates for bilirubin.     

Stopped-flow studies of spectral changes in bilirubin-human serum albumin following an alkaline pH jump and following binding of bilirubin

A stopped-flow technique was used to study the spectral changes occurring in bilirubin-albumin following a pH jump as well as following binding of bilirubin at 25 degrees C. The changes were studied in two wavelength ranges, 280-310 nm (tyrosine residues) and 400-510 nm (bound bilirubin). The changes were analyzed according to a scheme of consecutive unimolecular reactions. Spectral monitoring of a pH jump from 11.3 to 11.8 reveals that the bilirubin-albumin complex changes its structure in several steps. The UV absorption spectra show that 3.8 tyrosine residues ionize in the first step, 2.5 in the second, none in the third, and 0.8 in the fourth and following steps. The visible absorption spectrum of bound bilirubin changes in the second, third, and fourth steps. The bilirubin spectra of the different bilirubin-albumin complexes occurring in the transition show a common isosbestic point at 445 nm, indicating a change of the dihedral angle between the two bilirubin chromophores in a three-step reaction. It is suggested that 1 tyrosine residue is located close to the bilirubin site and is externalized in the second step. Bilirubin binding to albumin was monitored at two pH values, 11.3 and 11.8. At pH 11.3 the complex changes its structure in a three-step relaxation sequence. A change of the dihedral angle between the bilirubin chromophores can explain the spectral changes observed in the second and third relaxations. Protonation of 0.7 tyrosine residues occurs in the third relaxation, suggesting internalization of a tyrosine residue as a late consequence of bilirubin binding. At pH 11.8 a two-step relaxation sequence follows bilirubin binding. No tyrosine protonation occurs. Bilirubin is probably bound more superficially at pH 11.8 than at pH 11.3.     

A high-pressure, infrared spectroscopic study of the solvation of bilirubin in lipid bilayers

The location of bilirubin IXa in lipid bilayers of dimyristoylphosphatidylcholine or dioleoylphosphatidylcholine was studied by determining the effects of bilirubin on the infrared spectra of the lipids as a function of pressure. It was found for both bilayers that bilirubin intercalated into the polymethylene chain region of the bilayer, being located between the carbonyl region and the methylene group two carbons from the methyl terminus. Small amounts of bilirubin interacted with the choline region of dioleoylphosphatidylcholine. Lesser amounts interacted with the choline region of dimyristoylphosphatidylcholine. This difference between the two types of bilayers was attributed to the degradation of small amounts of bilirubin IXa to more polar isomers in the presence of dioleoylphosphatidylcholine. In dioleoyl- but not dimyristoylphosphatidylcholine, bilirubin interacted with the C = O region, probably indicating that bilirubin in the latter type of bilayer was intercalated into the polymethylene chains above and below the double bond. Bilirubin decreased the pressure required for the liquid-crystal to gel-phase transition in both bilayers at 28 degrees C. Bilirubin was not forced out of either bilayer at pressures as high as 20 kbar.     

Flurorometric study of the partition of bilirubin among blood components: basis for rapid microassays of bilirubin and bilirubin binding capacity in whole blood

Bilirubin binds to many sites in blood, the strongest binding being to a single site on albumin. Secondary sites on albumin, most sites on other plasma proteins, and sites on erythrocyte membranes have affinities for bilirubin that are at most one-hundredth as great. Bilirubin binds to hemoglobin in red cells with an effective affinity that is less than one-thousandth that of the primary albumin site. Essentially the only bilirubin present in blood which fluoresces is that bound to the primary albumin site. Almost all the other bilirubin in blood fluoresces with a yield no more than one-fiftieth as large. Quantitative fluorometry of whole blood is possible using the “front-face” technique. The concentration of bilirubin bound to the primary albumin site can be determined in this way. The albumin binding capacity of a blood specimen can be similarly assayed upon titration of the specimen with bilirubin. The nonionic detergent dodecyldimethylamine oxide (DDAO) scavenges bilirubin from all sites in blood, and, since bilirubin is fluorescent in DDAO micelles, the total blood bilirubin can be assayed fluorometrically after addition of DDAO to the specimen. This detergent method also allows facile assay of red-cell-bound bilirubin. These fluorometric assays for total blood bilirubin, albumin-bound bilirubin, and albumin binding capacity are simple and rapid and use very small volumes of blood. They should be of great value in the research on neonatal jaundice and in its clinical management.     

Triamcinolone activation of renal ammonia production.

The effects of scillaren and dinitrophenol on bilirubin excretion by the perfused rat liver were studied. Both compounds inhibited bile flow, scillaren by 20 to 40%, and dinitrophenol by 60 to 80%. Bilirubin excretion was also impaired. However, the effect of scillaren on bilirubin excretion was less than that on bile flow, as indicated by an increase in the bile bilirubin concentration, whereas dinitrophenol had a greater effect on bilirubin excretion than on bile flow. Dinitrophenol also inhibited the hepatic removal of unconjugated bilirubin from the perfusate, probably because it impaired the initial uptake and/or storage of unconjugated bilirubin by the perfused liver.     

A new tactic for the treatment of jaundice: an injectable polymer-conjugated Bilirubin oxidase

Bilirubin oxidase (BOX) derived from Myrothecium verrucaria was modified with polyethylene glycol (PEG). When the conjugated PEG-BOX was given intravenously to rats, its plasma half-life was 20 times longer than that of native BOX. In our preliminary investigations with experimentally jaundiced rats, the plasma bilirubin level dropped to normal after only one injection, and the low bilirubin level could be maintained for 12-48 hr; native BOX had a transitory suppressive effect that lasted only a few hours. The antigenicity of PEG-BOX was greatly reduced as expected. PEG-BOX appears to have potential value for the treatment of hyperbilirubinemia observed in such diseases as fulminant hepatitis and neonatal bilirubin encephalopathy.     

Quantitative determination of bilirubin by inhibition of chemiluminescence from lucigenin.

Bilirubin is a metabolic breakdown product of blood haem, of great biological and diagnostic importance. A new chemiluminescence (CL) method has been developed for the quantification of bilirubin. The method is combined with the flow injection analysis (FIA) technique and based on the inhibition effect of bilirubin on the CL from the lucigenin-hydrogen peroxide system in an alkaline medium. Under the optimum conditions, the decreased CL intensity was proportional to the concentration of bilirubin, in the range 0.0585-58.47 microg/mL. The detection limit estimated from the calibration graph was about 7.8826 ng/mL. The relative standard deviation (RSD) of 10 parallel measurements (1 x 10(-4) mol/L bilirubin) was 2.5%. Recoveries of bilirubin were found to fall in the range 94-97.5% using control sera. The method is interference-free, fast and easy to carry out.     

Inhibition of secretory phospholipase A2 enzyme by bilirubin: A new role as endogenous anti-inflammatory molecule

Bilirubin is a powerful antioxidant that suppresses the inflammatory process. However its interaction with proinflammatory PLA₂ enzyme is not known. Inhibition of several secretory phospholipase A₂ (sPLA₂) enzyme activities by bilirubin was studied using ¹⁴C-oleate labeled Escherichia coli as substrate. Bilirubin inhibits purified sPLA₂ enzyme from Vipera russellii and Naja naja venom and partially purified sPLA₂ enzymes from human ascitic fluid, pleural fluid and normal serum in a dose dependent manner. IC₅₀ values calculated for these enzymes ranges from 1.75 to 10.5 μM. Inflammatory human sPLA₂ enzymes are more sensitive to inhibition by bilirubin than snake venom sPLA₂s. Inhibition of sPLA₂ activity by bilirubin is independent of calcium concentration. Increasing substrate concentration (upto 180 nmol) did not relieve the inhibition of sPLA₂ by bilirubin and it is irreversible. Bilirubin quenched the relative fluorescence intensity of sPLA₂ in a dose dependent manner in the same concentration range at which in vitro sPLA₂ inhibition was observed. In the presence of bilirubin, apparent shift in the far UV-CD spectra of sPLA₂ was observed, indicating a direct interaction with the enzyme. Inhibition of sPLA₂ induced mouse paw edema by bilirubin confirms its sPLA₂ inhibitory activity in vivo also. These findings indicate that inhibition of sPLA₂ by bilirubin is mediated by direct interaction with the enzyme and bilirubin may act as an endogenous regulator of sPLA₂ enzyme activity.     

CIRCADIAN VARIABILITY OF BILIRUBIN IN HEALTHY MEN DURING NORMAL SLEEP AND AFTER AN ACUTE SHIFT OF SLEEP

Bilirubin is a laboratory test widely used for patient care, especially neonatal patients and patients with anemia or suspected liver disorders. Bilirubin has also been shown to be associated with sleep pattern and oxidative stress. The aim of this study was to investigate the variation of bilirubin in a group of healthy individuals with normal night sleep as well as during acutely displaced sleep, as sleep timing varies immensely between individuals while clinical samples are still mainly taken in the morning. We studied the diurnal variation of bilirubin during night-sleep and day-sleep conditions in seven healthy volunteers. Serum samples were collected every hour (50 samples/individual) to evaluate the effect of different sampling times and sleep displacement on the test results. The mean acrophases (peak time) occurred at 10.6?h during the night-sleep condition and at 18.4?h during the day-sleep condition. The diurnal intraindividual variation was high during both the night-sleep and day-sleep conditions, with coefficients of variation (CV) in the range of 12.8 to 42.5%. The diurnal variation was higher during the day compared to night-sleep condition. Thus, bilirubin sampling should be restricted to the morning, preferably after a normal night sleep, to minimize intraindividual variation. (Author correspondence: anders.larsson@akademiska.se)     

Bilirubin inhibits Plasmodium falciparum growth through the generation of reactive oxygen species

Free heme is very toxic because it generates highly reactive hydroxyl radicals ((.)OH) to cause oxidative damage. Detoxification of free heme by the heme oxygenase (HO) system is a very common phenomenon by which free heme is catabolized to form bilirubin as an end product. Interestingly, the malaria parasite, Plasmodium falciparum, lacks an HO system, but it forms hemozoin, mainly to detoxify free heme. Here, we report that bilirubin significantly induces oxidative stress in the parasite as evident from the increased formation of lipid peroxide, decrease in glutathione content, and increased formation of H(2)O(2) and (.)OH. Bilirubin can effectively inhibit hemozoin formation also. Furthermore, results indicate that bilirubin inhibits parasite growth and induces caspase-like protease activity, up-regulates the expression of apoptosis-related protein (Gene ID PFI0450c), and reduces the mitochondrial membrane potential. (.)OH scavengers such as mannitol, as well as the spin trap alpha-phenyl-n-tert-butylnitrone, effectively protect the parasite from bilirubin-induced oxidative stress and growth inhibition. These findings suggest that bilirubin, through the development of oxidative stress, induces P. falciparum cell death and that the malaria parasite lacks an HO system probably to protect itself from bilirubin-induced cell death as a second line of defense.     

A comprehensive review of bilirubin determination methods with special emphasis on biosensors

Bilirubin, is a tetrapyrrole yellow coloured compound found in digestive juice. It is generated from degradation of hemoglobin (Hb). The normal range of total bilirubin in serum is 0.30–1.20 mg/dl. The elevated range of serum bilirubin is considered as biomarker for finding and therapeutic administration of many liver diseases. Various analytical methods for determination of bilirubin, including spectrophotometery, thin layer chromatography, fluorometry, capillary electrophoresis, high performance liquid chromatographic, polarography and chemiluminescence have been applied for clinical purposes. These conventional methods are tedious, time-consuming, and require costly equipments and skilled person to operate. To overcome these limitations, the most popular biosensing technology has been employed at a large scale. The present review describes the principle, advantages and disadvantages of different analytic methods for measurement of bilirubin with focusing on biosensors, including electrochemical, photo-electrochemical, piezoelectric, optical and luminescent biosensors in detail. The working conditions for optimum activity and shelf life of all bilirubin biosensors have been summarized & compared and their future perspectives are discussed.     

The physiology of bilirubin: health and disease equilibrium

Bilirubin has several physiological functions, both beneficial and harmful. In addition to reactive oxygen species-scavenging activities, bilirubin has potent immunosuppressive effects associated with long-term pathophysiological sequelae. It has been recently recognized as a hormone with endocrine actions and interconnected effects on various cellular signaling pathways. Current studies show that bilirubin also decreases adiposity and prevents metabolic and cardiovascular diseases. All in all, the physiological importance of bilirubin is only now coming to light, and strategies for increasing plasma bilirubin levels to combat chronic diseases are starting to be considered. This review discusses the beneficial effects of increasing plasma bilirubin, incorporates emerging areas of bilirubin biology, and provides key concepts to advance the field.