Bilirubin Binding Research Articles

Application of phenol red as a marker ligand for bilirubin binding site at subdomain IIA on human serum albumin

The drug–bilirubin interaction for all drugs administered especially to infants with hyperbilirubinemia should be evaluated for their ability to displace bilirubin and vice versa. In order to examine whether phenol red (PhRed) can be used as a marker for bilirubin binding site located in subdomain IIA the interaction between PhRed and human serum albumin (HSA) in buffer solution or in normal and pathological sera solutions with different HSA:bilirubin molar ratio was investigated using absorption/absorption difference spectroscopy and molecular docking method. Six sulfonamides representing the binding site in the subdomain IIA and known to influence the binding of bilirubin were used for the PhRed displacement studies. The absorption spectra for PhRed completely bound to HSA showed significant differences in the spectral characteristic relative to the spectral profile of free PhRed. The intensity of the peak originating from the bivalent anionic form of dye was strongly reduced and the maximum peak position was red-shifted by 12nm. The binding constant (K) of the bivalent anionic form of PhRed, calculated from absorbance data, was 1.61·104Lmol−1. The variations of the absorption and absorption difference spectra of PhRed in the presence of HSA–bilirubin complex were indicative of the inhibition of PhRed binding process by bilirubin. Binding of PhRed carried out in the presence of sulfonamides showed that drugs and PhRed have a common site which also involves bilirubin. In agreement with the results of the spectroscopic analysis and molecular docking it was concluded that PhRed may be applied as a marker in the study of the binding of drugs to high-affinity bilirubin binding site.

Neuroprotective Core Measure 6: Protecting Skin - Neuroprotective Care in the Newborn: Does Skin Protect the Immature Brain From Hyperbilirubinemia?

Hyperbilirubinemia continues to pose a significant and common problem in the newborn period. Exposure of the brain to high levels of unconjugated bilirubin leads to acute bilirubin encephalopathy and kernicterus, especially in preterm infants. Given the shared embryological origin of the skin (epidermis) and brain, we hypothesized that cutaneous binding of unconjugated bilirubin to skin (i.e., jaundice) might protect the immature brain. Support for this hypothesis requires direct quantification of binding of unconjugated bilirubin to cutaneous structures. Bilirubin binding was tested using a series of in vitro experiments wherein newborn skin and vernix caseosa were exposed to physiologically-relevant solutions of bilirubin. Tissue binding was assessed spectrophotometrically and via bilirubin autofluorescence. Study findings indicate the following: (1) unconjugated bilirubin binds quickly and avidly to thin films of vernix caseosa; (2) bilirubin binds to human epidermis in vitro via a mechanism involving dermal diffusion; (3) unconjugated bilirubin localizes to the dermis and epidermis as shown by autofluorescence; and (4) topical application of vernix caseosa to the epidermis augments bilirubin binding; i.e., increases jaundice. These findings are consistent with a physiological neuroprotective role for the skin in shielding the immature brain from high levels of unconjugated bilirubin. New therapies based on these results are envisioned with the goal of increasing cutaneous bilirubin binding (jaundice) thereby protecting the developing brain and facilitating bilirubin excretion with phototherapy.

Bilirubin, model membranes and serum albumin interaction: The influence of fatty acids

Electronic circular dichroism (ECD), absorption and fluorescence spectroscopy were used to study the enantioselective interactions which involved bilirubin (BR), liposomes, human serum albumin of two different purities, pure (HSA) and non-purified of fatty acids (FA-HSA), and individual fatty acids.The application of the ECD technique to such a complex problem provided a new perspective on the BR binding to liposomes. Our results demonstrated that in the presence of pure HSA, BR preferred to bind to the protein over the liposomes. However, in the presence of FA-HSA, BR significantly bound to the liposomes composed either of DMPC or of sphingomyelin and bound only moderately to the primary and secondary binding sites of FA-HSA even at high BR concentrations. For the DMPC liposomes, even a change of BR conformation upon binding to the primary binding site was observed. The individual saturated fatty acids influenced the BR binding to HSA and liposomes in a similar way as fatty acids from FA-HSA. The unsaturated fatty acids interacted with BR alone and prevented it from interacting with either 99-HSA or the liposomes. In the presence of arachidonic acid, BR interacted enantioselectively with the liposomes and only moderately with 99-HSA.Hence, our results show a substantial impact of the liposomes on the BR binding to HSA. As a consequence of the existence of fatty acids in the blood plasma and in the natural structure of HSA, BR may possibly bind to the cell membranes even though it is normally bound to HSA.

Risk of BIND and kernicterus in late preterm

Severe hyperbilirubinemia can induce devastating and permanent neurodevelopmental handicaps in infants. The occurrence of hyperbilirubinemia is higher in late preterm than in term infants, ranging from 8 to 40% of the formers according to different definitions of hyperbilirubinemia. The mechanisms by which this occurs is not completely understood, however an increased bilirubin load on hepatocyte, as result of decreased erythrocyte survival and increased erythrocyte volume, increased enterohepatic circulation of bilirubin, decreased hepatic uptake of bilirubin from plasma, defective bilirubin conjugation, and diminished serum bilirubin binding capacity play a relevant role. Hyperbilirubinemia in late preterm infants is not only more prevalent than in term neonates, but also it occurs later and is more severe and protracted. In fact, Maisels et al. (Pediatrics 2006;117:1169) demonstrated that at 72 hours of life the value of 50° percentile of trans-cutaneous bilirubin (TcB) is 9 mg/dl in 35-37 wks infants, while is 40 wks infants. Late preterm infants are at higher risk of bilirubin induced neurological dysfunction (BIND) and kernicterus than term infants. The mechanisms that potentially could explain the high susceptibility of central nervous system to bilirubin-induced damage in late preterm neonates have not been well defined. However, some of the factors that can potentially contribute are the diminished serum bilirubin binding capacity due to the lower serum albumin levels, an enhanced permeability of the blood–brain barrier to unconjugated bilirubin influx, and an immaturity of neuronal protective mechanisms. This is probably the reason why late preterm neonates are at an increased risk to develop acute bilirubin encephalopathy and or kernicterus as demonstrated in the USA pilot kernicterus registry (Semin Perinatol 2006;30:89) in which this category of infants is over-represented compared to term neonates. Thus, clinicians need to be more concerned and conscientious to identify late preterm’s risk for severe hyperbilirubinemia in view of their increased susceptibility to BIND. This can allow of planning a prevention program including nursing and parental education, screening for jaundice in the nursery, the provision of lactation support, timely post discharge follow-up, and appropriate treatment when clinically indicated.

Bilirubin Binding with Liver Cystatin Induced Structural and Functional Changes

Cysteine proteinases and their inhibitors play a significant role in the proteolytic environment of the cells. Inhibitors of cysteine proteinases regulate the activity of these enzymes helping in checking the degdration activity of cathepsins. The bilirubin secreated by liver cells can bind to cystatin present in the liver resulting in its functional inactivation, which may further lead to the increase in cathepsins level causing liver cirrhosis. In case of some pathophysiological conditions excess bilirubin gets accumulated e.g. in presence of Fasciola hepatica (liver fluke) in mammals and humans, leading to liver cirrhosis and possibly jaundice or normal blockade of bile duct causing increased level of bilirubin in blood. Protease-cystatin imbalance causes disease progression. In the present study, Bilirubin (BR) and liver cystatin interaction was studied to explore the cystatin inactivation and structural alteration. The binding interaction was studied by UV-absorption, FT-IR and fluorescence spectroscopy. The quenching of protein fluorescence confirmed the binding of BR with buffalo liver cystatin (BLC). Stern-Volmer analysis of BR-BLC system indicates the presence of static component in the quenching mechanism and the number of binding sites to be close to 1. The fluorescence data proved that the fluorescence quenching of liver cystatin by BR was the result of BR-cystatin complex formation. FTIR analysis of BR-Cystatin complex revealed change in the secondary structure due to perturbation in the microenvironment further confirmed by the decreased caseinolytic activity of BLC against papain. Fluorescence measurements also revealed quenching of fluorescence and shift in peak at different time intervals and at varying pH values. Photo-illumination of BR-cystatin complex causes change in the surrounding environment of liver cystatin as indicated by red-shift. The binding constant for BR-BLC complex was found to be 9.279 × 10(4) M(-1). The cystatin binding with bilirubin has a significant biophysical and pathophysiological significance, hence our effort to study the same.

Polyacrylate guanidine and polymethacrylate guanidine as novel cationic polymers for effective bilirubin binding

The effect of polycationic polymers of polyacrylate guanidine (PAG) and polymethacrylate guanidine (PMAG) on bilirubin absorbance were studied in phosphate buffer (pH 7.4). It was shown that the change in absorbance spectra of bilirubin in the presence of PAG/PMAG can be associated with the formation of a bilirubin-polymer complex and dissociation of tetramers on bilirubin monomers. Also, the organic-inorganic composite materials based on silica gels and guanidine polymers were synthesized via the sol-gel technique. The incorporated guanidine polymers have a big influence on particle size distribution of silica gel due to their high cross-linking ability. The infrared spectroscopy revealed the presence of guanidine polymers inside solid networks of silica gel. The bilirubin adsorption process onto a guanidine functionalized silica surface was investigated. The Langmuir and Redlich-Peterson isotherm models were tested to explain the adsorption mechanism. The analysis of the adsorption isotherms confirms the possibility of electrostatic interactions of bilirubin molecules with guanidine polymers incorporated inside silica matrix. We conclude that cationic guanidine polymers might be effectively applied for bilirubin removal.

A microscopic evaluation of collagen–bilirubin interactions: in vitro surface phenomenon

This study is carried out to understand the morphology variations of collagen I matrices influenced by bilirubin. The characteristics of bilirubin interaction with collagen ascertained using various techniques like XRD, CLSM, fluorescence, SEM and AFM. These techniques are used to understand the distribution, expression and colocalization patterns of collagen-bilirubin complexes. The present investigation mimic the in vivo mechanisms created during the disorder condition like jaundice. Fluorescence technique elucidates the crucial role played by bilirubin deposition and interaction during collagen organization. Influence of bilirubin during collagen fibrillogenesis and banding patterns are clearly visualize using SEM. As a result, collagen-bilirubin complex provides different reconstructed patterns because of the influence of bilirubin concentration. Selectivity, specificity and spatial organization of collagen-bilirubin are determined through AFM imaging. Consequently, it is observed that the morphology and quantity of the bilirubin binding to collagen varied by the concentrations and the adsorption rate in protein solutions. Microscopic studies of collagen-bilirubin interaction confirms that bilirubin influence the fibrillogenesis and alter the rate of collagen organization depending on the bilirubin concentration. This knowledge helps to develop a novel drug to inhibit the interface point of interaction between collagen and bilirubin.

Glutathione transferase-A2 S112T polymorphism predicts survival, transplant-related mortality, busulfan and bilirubin blood levels after allogeneic stem cell transplantation

Busulfan liver metabolism depends on glutathione, a crucial mediator of cellular and systemic stress. Here we investigated 40 polymorphisms at 27 loci involved in hepatic glutathione homeostasis, with the aim of testing their impact on the clinical outcome of 185 busulfan-conditioned allogeneic transplants. GSTA2 S112T serine allele homozygosity is an independent prognostic factor for poorer survival (RR=2.388), for increased any time- and 100-day transplant-related mortality (RR=4.912 and RR=5.185, respectively). The genotype also predicts a wider busulfan area under the concentration-time curve (1214.36 ± 570.06 vs. 838.10 ± 282.40 mMol*min) and higher post-transplant bilirubin serum levels (3.280 ± 0.422 vs. 1.874+0.197 mg/dL). In vitro, busulfan elicits pro-inflammatory activation (increased NF-KappaB activity and interleukin-8 expression) in human hepatoma cells. At the same time, the drug down-regulates a variety of genes involved in bilirubin liver clearance: constitutive androstane receptor, multidrug resistance-associated protein, solute carrier organic anion transporters, and even GSTA2. It is worthy of note that GSTA2 also acts as an intra-hepatic bilirubin binding protein. These data underline the prognostic value of GSTA2 genetic variability in busulfan-conditioned allotransplants and suggest a patho-physiological model in which busulfan-induced inflammation leads to the impairment of post-transplant bilirubin metabolism.

Raltegravir In Vitro Effect on Bilirubin Binding

Drugs that displace bilirubin from albumin may increase the risk of kernicterus in neonates. We evaluated the effect of raltegravir on bilirubin-albumin binding in pooled neonatal serum using the peroxidase method. Raltegravir had minimal effect on bilirubin-albumin binding at concentrations of 5 and 10 µM, caused a small but statistically significant increase in unbound bilirubin at 100 µM and caused potentially harmful increases at 500 and 1000 µM. Our data suggest that the effect of raltegravir on neonatal bilirubin binding is unlikely to be clinically significant at typical peak concentrations reached with usual dosing.

Studies on the chemico-biological characteristics of bilirubin binding with collagen

The clinical impact of bilirubin on collagen is investigated using various physical, chemical and biological methods. Thermo gravimetric analysis and differential scanning analysis of collagen–bilirubin complex matrices indicate that crosslinking does not alter their thermal behavior of collagen. The polydispersity of collagen–bilirubin complex increases in the reacting medium suggesting that there is an increase in the number of interacting points between them. Based on the zeta potential values, the rate of mobility of interacted complex decreases by inferring the extent of binding compared to the control collagen. Emission intensity begins to increase with increase in concentration of bilirubin which ascribes the conformational changes around the aromatic amino acids in collagen. Binding is indicated by an increase in resonance units and the responses are corrected by subtraction of those obtained for native collagen. Bilirubin showed a higher affinity for collagen at a concentration of about 25nM/mg. In this study, the association rate has been calculated which depicts the increased affinity of bilirubin to collagen. Affinity for bilirubin to collagen has been found to be 8.89×10−3s−1. The greater part of binding of bilirubin to collagen is found to be electrostatic in nature. The investigation leads to comprehend the affinity of collagen–bilirubin complex during jaundice diseased tissues.

Studies on the molecular significance in the interaction of bilirubin with collagen

The present investigation is aimed to understand the physiological significance of bilirubin interaction with collagen. In human skin, collagen absorbs both free bilirubin and serum bound bilirubin from the human system. Interaction between bilirubin and collagen depends on time, temperature and concentration of bilirubin. There is an increase in the aggregation rate of collagen in the presence of biliruibin. At physiological condition, 125nM of bilirubin is the maximum concentration absorbed by per mg of collagen molecule. Bilirubin accelerates the lateral growth of collagen fibrils by shifting its rate of nucleation. Moreover, collagen–bilirubin complex exhibit a tendency to undergo adsorption onto the surface of the fibroblast cells, showing detrimental effects on fibroblasts proliferations. Based on the collagen binding assays, the binding of bilirubin to collagen is found to be electrostatic in nature, which confirms binding between the amino acid fragment of α1 (I) region of collagen and carboxyl group of bilirubin. The biotinylated bilirubin derivatives show better binding to α1 (I) chain rather than α2 (I) chains which clearly designates that bilirubin shows greater affinity to α1 chains of collagen. This novel approach directs to reduce the occurrence of bilirubin in hyperbilirubinemia patients.

Improvement of bilirubin adsorption capacity of cellulose acetate/polyethyleneimine membrane using sodium deoxycholate

Bilirubin (BR) adsorbents have low removal efficiency because of the tight binding of BR with human albumin (HA) in a complicated blood system. Sodium deoxycholate (SDC) was selected as an adsorption promoter to improve the BR adsorption capacity of a cellulose acetate (CA)/polyethyleneimine (PEI) membrane. Static adsorption experiments show that the maximum BR adsorption capacity of the membrane with SDC in BR–HA mock solution is 100–200% at the molar ratio of SDC to HA ranging from 8 to 12, higher than that without SDC. It is also found that SDC is more efficiently adsorbed by the membrane than BR and HA. Absorption, circular dichroism, and zeta potential studies demonstrate that SDC can be bound with the BR–HA complex to form a ternary BR–HA-SDCm complex. On the basis, the facilitated adsorption mechanism of BR with SDC was proposed that SDC aggregates or micelles form a quasi-multilayer adsorption on the membrane, increase approachable binding sites, and prolong the distance between the BR–HA complex and the membrane. Thus, SDC as a spacer reduces the influence of the steric hindrance of HA, resulting in an enhanced BR adsorption capacity. Dynamic adsorption results further evidence the facilitated adsorption mechanism.

Identification of Bilirubin Binding Site in Type I Collagen

The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α1(I) chain but not to α2(I) chains which clearly designates that bilirubin shows greater affinity to α1 chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible.

Albumin domain II mutant with high bilirubin binding affinity has a great potential as serum bilirubin excretion enhancer for hyperbilirubinemia treatment

Background4Z,15Z-bilirubin-IXα (BR), an endogenous toxic compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. Our previous findings suggest that both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of BR, and especially Lys199 in stand-alone domain II plays a prominent role in the renal elimination of BR. Our hypothesis is that HSA-domain II with high BR binding would be a useful therapeutic agent to treat hyperbilirubinemia in patients with impaired liver function. MethodsUnbound BR concentrations were determined using a modified HRP assay. To evaluate the effect of pan3_3-13 domain II mutant in promoting urinary BR excretion, the serum concentration and urinary excretion amount of BR were determined using bile duct ligation mice. ResultsAfter three or six rounds of panning, pan3_3-13 and pan6_4 were found to have a significantly higher affinity for BR than wild-type domain II. Administration of pan3_3-13 significantly reduced serum BR level and increased its urinary excretion in the disease model mice as compared to wild-type domain II treatment. ConclusionsThese results suggest that pan3_3-13 has great potential as a therapeutic agent that promotes urinary BR excretion in hyperbilirubinemia. General significanceThis is the first study to be applied to other HSA bound toxic compounds that are responsible for the progression of disease, thereby paving the way for the development of non-invasive and cost effective blood purification treatment methods.

Orphans in the world of drugs

Orphans in the world of drugs

356 Albumin Synthesis Rates in VLBW Infants - Effects of High dose Amino Acid and Lipid Administration from Birth Onwards

IntroductionAlbumin is one of the most important proteins in plasma and plays a key role in physiological processes like preservation of colloid osmotic pressure and binding of bilirubin and drugs....

5-Imino-1,2-4-thiadiazoles and quinazolines derivatives as glycogen synthase kinase 3β (GSK-3β) and phosphodiesterase 7 (PDE7) inhibitors: Determination of blood–brain barrier penetration and binding to human serum albumin

5-Imino-1,2,4-thiadiazoles and quinazolines derivatives as glycogen synthase kinase 3β (GSK-3β) and phosphodiesterase 7 (PDE7) inhibitors were characterized for their ability to pass the blood–brain barrier (BBB) together with their human serum albumin (HSA) binding using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). To study the blood–brain barrier penetration, a parallel artificial membrane permeability assay (PAMPA) using a porcine brain lipid was employed. For the HPLAC investigation, HSA was previously covalently immobilized to the silica matrix of the HPLC column. This HSA-based column was used to characterize the high affinity binding sites of 5-imino-1,2,4-thiadiazoles and quinazolines derivatives to HSA. Displacement experiments in the presence of increasing concentrations of competitors known to bind selectively to the main binding sites of HSA were carried out to determine their possible binding site. The same drug–protein system was studied by CD.The analysed compounds were able to pass BBB, they present good drug-like properties and they showed a high affinity to HSA. Competition experiments showed an anticooperative interaction at sites I and II, and an independent binding at bilirubin binding site on HSA.

A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin

11-(Dansylamino) undecanoic acid (DAUDA) is a dansyl-type fluorophore and has widely used as a probe to determine the binding site for human serum albumin (HSA). Here, we reported that structure of HSA-Myristate-DAUDA ternary complex and identified clearly the presence of two DAUDA molecules at fatty acid (FA) binding site 6 and 7 of HSA, thus showing these two sites are weak FA binding sites. This result also show that DAUDA is an appropriate probe for FA site 6 and 7 on HSA as previous studied, but not a good probe of FA binding site 1 that is likely bilirubin binding site on HSA.

Bilirubin Binding Contributes to the Increase in Total Bilirubin Concentration in Newborns with Jaundice

Bilirubin Binding Contributes to the Increase in Total Bilirubin Concentration in Newborns with Jaundice

Hypoalbuminemia following Abdominal Surgery Leads to High Serum Unbound Bilirubin Concentrations in Newborns Soon after Birth

Background: The serum concentration of unbound bilirubin (UB), which is bilirubin not bound to albumin (Alb), is a better index than total bilirubin concentration (TB) for identifying infants at risk for developing bilirubin neurotoxicity. The degree to which the hypoalbuminemia following abdominal surgery in jaundiced newborns affects bilirubin binding is unknown. Objective: To determine whether lower Alb occurring in newborns undergoing abdominal surgery shortly after birth results in significantly higher UB in serum versus nonsurgical patients at comparable serum TB. Methods: A matched case-control study was conducted with term and late-preterm newborns. The surgery group included 15 newborns who underwent abdominal operation within 3 days after birth. Clinical and laboratory data (serum UB, TB, and Alb concentrations, UB/TB ratio, and binding constant) in the surgery group were collected and compared with those of 30 control newborns who did not undergo abdominal surgery (control group). Results: Serum UB and the UB/TB ratio in the surgery group were significantly higher than those in the control group (p < 0.02, p < 0.001, respectively), whereas there were no significant differences in serum TB and binding constant between the groups. Serum Alb concentrations in the surgery group were significantly lower than those in the control group (p < 0.001). When pre- and postoperative serum Alb concentrations were compared, there was a significant decrease from 3.4 to 2.7 g/dl (p < 0.001). Conclusions: Our study suggests that hypoalbuminemia following abdominal surgery causes a higher serum UB at comparable serum TB in newborns.