Bilirubin Binding Capacity Research Articles

Synergistic effect of chlorogenic acid and vitamin D3 (cholecalciferol) on in-vitro glycation may assist in prevention of Polycystic Ovarian Syndrome (PCOS) progression - A biophysical, biochemical and in-silico study

Advanced glycation end products (AGEs) are formed through non-enzymatic glycation, that have been linked to various diseases, including polycystic ovarian syndrome (PCOS) playing a critical role leading to secondary comorbidities such as diabetes-related problems, cardiovascular complications, infertility, etc. As a result, there has been a lot of research into AGE-inhibiting phytochemicals for the remediation and obstruct progression of glycation-related illnesses. The current study is based on in-vitro protein model, in which human serum albumin have been used to investigate the cumulative effect of chlorogenic acid (CGA) and cholecalciferol (vitamin D) on glycation and evaluate their inhibitory impact on AGEs production in the presence of methylglyoxal. Through the application of several biochemical and biophysical techniques, we were able to examine the synergistic impact of both the compounds on albumin structure and its biochemical properties during different stages of glycation. According to Nitro-blue tetrazolium assay results indicate that CGA and vitamin D inhibited fructosamine (early glycation product) production. Moreover, free thiol and lysine residues were significantly increased whereas protein carbonyl levels were significantly decreased. Additive effect of CGA and vitamin D were associated with reduced AGEs fluorescence and increased tryptophan and tyrosine fluorescence. Amadori-albumin after treatment showed some evidence of regaining its alpha-helicity as measured by far-UV CD spectrum. Furthermore, secondary structural alterations were confirmed by Fourier transform infrared spectroscopy (FTIR). ANS (1-anilinonaphthalene-8-sulfonic acid) fluorescence spectra also displayed less revelation of hydrophobic patches. Bilirubin binding capacity was also restored which showed functional recovery of HSA. The electrophoretic mobility was also restored which is portrayed by SDS-PAGE. Additionally, to predict the anti-aggregation potential of CGA and vitamin D, congo red assay and ThT fluorescence was performed which reveal low aggregate formation after treatment. These results corroborated with scanning electron microscopy and confocal microscopy. Docking and simulation results also reveal spontaneous binding of CGA and vitamin D on subdomain IIA of HSA favoring their binding thermodynamically. All the findings suggest that chlorogenic acid and cholecalciferol given in combination might help in prevention of PCOS progression and its related complications.

Bilirubin/Albumin Levels in Patients with Alzheimer's Disease and Use of Intravenous Albumin for Its Treatment

Background: Bilirubin may be an upstream activator inducing Alzheimer's disease (AD). Methods: Bilirubin/albumin (ALB) levels in dementia patients with Aβ deposition were explored. Human albumin at a dose of 10 g every 2 weeks for 24 weeks was prescribed to AD patients to examine the effect of albumin on AD symptoms. Findings: Significantly higher indirect bilirubin (IBIL) levels and higher ratio of IBIL to albumin (IBIL/ALB) were reported in dementia patients with Aβ deposition, including AD, dementia with Lewy bodies and general paresis of insane.Intravenous albumin significantly lowered the ratio of IBIL to albumin (IBIL/ALB) at week 24. Analysis of the combined data of the entire 28 weeks of assessment period using the area under curve show significantly improvements in change of Alzheimer's Disease Cooperative Study Activities of Daily Living Inventory scale scores and Clinical Dementia Rating Scale-Sum of Boxes scores. Interpretation: IBIL and IBIL/ALB was significantly higher in dementia patients with Aβ deposition. Bilirubin may be an upstream activator that induces AD. Consistent with these clinical findings, our intervention study shows that increasing serum bilirubin binding capacity from albumin by intravenous supplementation of human albumin yields benefit on daily function and dementia severity in AD patients. Targeting IBIL/ALB has highly potential for AD treatment. Trial Registration: The study was registered at chictr.org.cn (ChiCTR-IOR-17011539). Funding Statement: This work was supported by the National Funds for Developing Local Colleges and Universities (B16056001), Natural Science Foundation research team of Guangdong Province (2018B030312001) (to J.L), and Guangdong Natural Science Foundation (2017A030313662), Science and Technology Program of Guangzhou (201707010046) (to C.Z). Declaration of Interests: All authors declare that there are no conflicts of interest. Ethics Approval Statement: The Ethics Committee of the Affiliated Brain Hospital of Guangzhou Medical University (Guangzhou Huiai Hospital) approved this study. A written informed consent was obtained from the participants or their nearest relatives.

Pioglitazone inhibits advanced glycation induced protein modifications and down-regulates expression of RAGE and NF-κB in renal cells

The present work aims to determine the effect of pioglitazone on in-vitro albumin glycation and AGE-RAGE induced oxidative stress and inflammation. Bovine serum albumin was glycated by methylglyoxal in absence or presence of pioglitazone. Glycation markers (fructosamine, carbonyl groups, β-amyloid aggregation, thiol groups, bilirubin binding capacity and AOPP); protein conformational changes (native-PAGE and HPLC analysis) were determined. Cellular study was done by estimating antioxidants, ROS levels, expression profile of membrane RAGE, NF-κB and levels of inflammatory cytokines (IL-6, TNF-α) using HEK-293 cell line. We observed that levels of glycation markers were reduced at higher concentration of pioglitazone as compared to glycated albumin. Structural analysis of glycated albumin showed inhibition of protein migration and structural changes when treated with pioglitazone. Pioglitazone has potentially restored cellular antioxidants and reduced levels of IL-6 and TNF-α by declining expression of membrane RAGE and NF-κB. In conclusion, pioglitazone preferentially binds to protein and alleviates protein structural changes by maintaining its integrity. Additionally, it suppresses RAGE and NF-κB levels hence alleviate cellular oxidative stress and inflammation.

Jack Aviv and brains of children.

Both lead intoxication in early childhood and deficient bilirubin-binding capacity (BBC) of blood in jaundiced neonates indicate risk for brain damage. Zinc protoporphyrin (ZPP) is a biomarker for lead intoxication (PbI) as well as well as for iron deficiency. Under the leadership of Jack Aviv, Aviv Biomedical, Inc. developed robust hematofluorometers for point-of-care assays of ZPP in blood and for the high-affinity BBC of blood. These assays use just drops of whole blood and are simple, fast and inexpensive. ZPP by hematofluorometry has been used world-wide as a primary screen for lead intoxication since 1979. Recent clinical studies enabled by an Aviv Biomedical, Inc. bilirubin hematofluorometer have renewed interest in BBC-based assessment of neurotoxicity for improved management of neonatal jaundice. This article sketches Jack Aviv's contribution to the development and application of hematofluorometry.

The Bilirubin Binding Panel: A Henderson-Hasselbalch Approach to Neonatal Hyperbilirubinemia.

Poor plasma bilirubin binding increases the risk of bilirubin neurotoxicity in newborns with hyperbilirubinemia. New laboratory tests may soon make it possible to obtain a complete bilirubin binding panel when evaluating these babies. The 3 measured components of the panel are the plasma total bilirubin concentration (BTotal), which is currently used to guide clinical care; the bilirubin binding capacity (BBC); and the concentration of non-albumin bound or free bilirubin (BFree). The fourth component is the bilirubin-albumin equilibrium dissociation constant, KD, which is calculated from BTotal, BBC, and BFree The bilirubin binding panel is comparable to the panel of components used in the Henderson-Hasselbalch approach to acid-base assessment. Bilirubin binding population parameters (not prospective studies to determine whether the new bilirubin binding panel components are better predictors of bilirubin neurotoxicity than BTotal) are needed to expedite the clinical use of bilirubin binding. At any BTotal, the BFree and the relative risk of bilirubin neurotoxicity increase as the KD/BBC ratio increases (ie, bilirubin binding worsens). Comparing the KD/BBC ratio of newborns with BTotal of concern with that typical for the population helps determine whether the risk of bilirubin neurotoxicity varies significantly from the inherent risk at that BTotal Furthermore, the bilirubin binding panel individualizes care because it helps to determine how aggressive intervention should be at any BTotal, irrespective of whether it is above or below established BTotal guidelines. The bilirubin binding panel may reduce anxiety, costs, unnecessary treatment, and the likelihood of undetected bilirubin neurotoxicity.

International Perspectives: Clinical Kernicterus in Preterm Infants in Japan

In recent years, clinical kernicterus has been diagnosed in preterm infants on the basis of motor (kinetic) disorders such as choreoathetosis and dystonia, impairment in auditory neuropathy-type hearing, and abnormal pallidal signals on head magnetic resonance imaging. In Japan, it is currently estimated that at least 8 to 9 cases of kernicterus (approximately 2 per 1,000 cases) occur annually in preterm infants of less than 30 weeks’ gestation. Therefore, there is an urgent need to revise our jaundice management approach in preterm infants. Two major characteristics of Japanese preterm infants who develop clinical kernicterus are: (1) a peak total serum/plasma bilirubin (TB) level at 2 weeks of age or later; and (2) no markedly high total bilirubinemia. Some patients without high TB levels have high levels of unbound serum/plasma bilirubin (UB). Consequently, we propose that in Japanese preterm infants, continuous monitoring of bilirubin (using transcutaneous bilirubinometry or direct TB measurements) and/or binding status (UB levels or bilirubin-binding capacity) may be necessary.

Albumin administration prevents neurological damage and death in a mouse model of severe neonatal hyperbilirubinemia.

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.

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 adsorption properties of water-soluble adsorbents with different cyclodextrin cavities in plasma dialysis system

In this study, we explored the use of α-, β- or γ-cyclodextrin (CD)-grafted polyethyleneimine (PEI) as water-soluble adsorbent for removing excess plasma bilirubin. To evaluate the bilirubin-binding capacity of these adsorbents, bovine serum albumin (BSA) solution or plasma with high level of bilirubin were dialyzed against CD-PEI-spiked dialysate. In BSA solution with an initial biliurbin concentration of 171.5 mg/L, α-CD-PEI, β-CD-PEI and γ-CD-PEI achieved adsorption capacities of 2.5, 5.8 and 3.8 mg/g, respectively. In a plasma dialysis system, 45.6% of bilirubin (260 mg/L) was removed from 200 mL plasma by 1 L dialysate spiked with 10 mg/mL β-CD-PEI, which was significantly higher than that removed by the same volume of BSA-spiked dialysate ( P < 0.05), demonstrating the strong bilirubin-binding ability of β-CD-PEI. The key feature of bilirubin adsorption was related to the CD functional group, not the PEI matrix. Subsequent molecular docking study indicated that the size of CD cavity could affect the affinity energy of CD–bilirubin complex. The cavity of β-CD was most suitable for accommodating the pyrrole rings of bilirubin. The inclusion complex of bilirubin and β-CD in the molar ratio of 1:2 was more logical in terms of affinity energy. All the results demonstrated the potential of β-CD-PEI (water-soluble adsorbent) as an effective agent for removing of bilirubin from plasma in dialysis system.

Determination of free bilirubin and its binding capacity by HSA using a microfluidic chip‐capillary electrophoresis device with a multi‐segment circular‐ferrofluid‐driven micromixing injection

A PMMA microfluidic chip-CE device with a multi-segment circular-ferrofluid-driven micromixing injector has been developed for the determination of free bilirubin and its binding capacity by HSA at equilibrium. The design of the device and its fabrication by a low cost CO(2) laser are discussed for intended applications. Under optimized conditions, the total binding capacity of HSA for bilirubin was determined as 16.3±1.4 mg/l00 mL human serum (n=3) and residual binding capacity for bilirubin 9.8 mg/100 mL (n=3) in normal infants. To assess risk of hyperbilirubinemia, free bilirubin and residual binding capacity by HSA provide a better indicator than total bilirubin, as neonates with impaired bilirubin binding capacity could be detected. In addition, residual binding capacity provides an advanced indicator to predict the onset of hyperbilirubinemia before the appearance of free bilirubin. HSA down to 94 nL is used in each titration and a full assay of four titrations takes up 376 nL HSA, sufficient for newborns with HSA in microliter range. The device has shown capable to provide adequate margin of protection to detect an early rising level of bilirubin and impaired binding capacity prior to the onset of jaundice condition.

Commentary on the bilirubin supplement

Neonatal jaundice is a normal transitional phenomenon after birth, as the newborn mammalian fetus adapts to life outside the womb. Hyperbilirubinemia in the neonate may even have a physiological role in the defense against oxidative stressors, such as oxygen, light and microbe-induced inflammationFall unavoidable in life on our planet. However, some babies have excessive elevations of bilirubin in their blood, and if they exceed the bilirubin-binding capacity of the circulation, mainly determined by albumin, bilirubin can accumulate in other tissues. One tissue that seems to be especially vulnerable is the brain with discrete areas prone to bilirubin-induced injury, for example, the globus pallidus, hippocampus, internal capsule, thalamus and eighth cranial nerve (VIII). The explanation for these regional susceptibilities and the mechanism of the toxicity have so far eluded researchers, although the fact that bilirubin can be toxic under some conditions encountered in the newborn is indisputable. Intriguing hypotheses have been proposed (Brites et al., this issue on pp S8–S13) and converging lines of investigations may eventually yield answers that might inform preventive strategies. One of the most tantalizing issue, perhaps because of its paradoxical character, is that bilirubin, an antioxidant in the usual biological circumstance, can cause or exacerbate oxidation under pathological conditions, such as excessive accumulation in tissues, triggering the production of cytokines, which, depending upon the timing in development and other confounding inflammatory states, such as infection, can alter neuronogenesis, cell-to-cell interactions or even contribute to abnormal apoptosis. What is clear to clinicians is that ‘bad things can happen’ in association with hyperbilirubinemia, even in apparently healthy term infants, if the bilirubin level is high enough (Watchko et al., this issue on pp S14–S19). Recent data suggest that much lower levels can also be associated with harm, such as neurodevelopmental impairment, profound impairment, cerebral palsy and hearing deficits, in the most immature neonates. Nonetheless, a blood bilirubin level, in and of itself, is rarely sufficient to predict toxicity in the individual case or even in a population. Thus, scientists remain focused on trying to predict which infants are most likely to develop excessive bilirubin accumulation in tissues outside the circulation, a phenomenon dependent upon more than the absolute level per se, but also upon albumin-binding capacity and affinity, the unbound or ‘free’ bilirubin (Bf) as well as the transporters that can facilitate bilirubin’s movement out of the brain. Excessive bilirubin production, as in the case of hemolysis (from any cause), is the main driver of load on the system and can often overload it. It is not surprising that hemolysis has been associated empirically with an increased risk of bilirubin-induced injury. However, impaired conjugation such as that associated with Gilbert’s disease can further exacerbate the load, contributing to more Bf, capable of entering the brain tissue. The genetic polymorphisms contributing to overload situations (that is, HO-1 overexpression or uridine diphosphoglucuronate glucuronosyltransferase deficiency) only exacerbate natural exigencies, such as prematurity, infection or acquired causes of heme degradation. Although these topics are not the focus of this supplement, one of the articles does attempt to model using Gunn rat pups the central nervous system’s Bf, probably a critical number to know if we could in the clinical setting (Watchko et al., this issue on pp S14–S19). These data support the contention that it is, indeed, the Bf in the CNS that is toxic. Still, clinicians are left with the rough empirical correlation of toxicity with the blood bilirubin level (in the case of the model 35 mg per 100 ml or 599 mmol l ), and the need to avoid such high levels. Therefore, until such time as the scientists can provide clinicians with more targeted diagnostics and therapeutics, the focus must necessarily be on prevalent practices. Moreover, the supplement senses this purpose well, dealing with safer management of newborn jaundice to prevent severe neonatal hyperbilirubinemia (Bhutani and Johnson, this issue on pp S4–S7), including a report on the Pilot USA Kernicterus Registry (Bhutani et al., this issue on pp S25–S45), the answering of frequently asked questions (Bhutani and Johnson, this issue on pp S20–S24), the presentation of a six-step strategy to prevent severe neonatal hyperbilirubinemia (Bhutani and Johnson, this issue on pp S61–S67), a description of a system-based approach to newborn care that could reduce the risk for severe jaundice (Stark, this issue on pp S53–S57), an argument for glucose-6-phosphate dehydrogenase deficiency screening (Kaplan and Hammerman, this issue on pp S46–S52) and a demonstration project for hyperbilirubinemia management by the Hospital Corporation of America (HCA) (Lazarus and Avchen, this issue on pp S58–S60). Correspondence: Dr DK Stevenson, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA. E-mail: dstevenson@stanford.edu Journal of Perinatology (2009) 29, S2–S3 r 2009 Nature Publishing Group All rights reserved. 0743-8346/09 $32

Bilirubin-binding Capacity in Premature Infants

To the Editor. — The recent article by Bender et al1 uses the “bilirubin binding capacity” to assess changes in plasma bilirubin binding associated with gestation and clinical status. They define binding capacity as the total bilirubin concentration (TBC) at which the “high affinity” (clinically important) albumin binding sites are “saturated” with bilirubin. The implication is that the risk of bilirubin neurotoxicity will increase significantly at or beyond this TBC. Because binding capacity is defined as the concentration of bound ligand at infinite ligand concentration,2 …

Bilirubin-binding Capacity in Low Birthweight Premature Infants

To the Editor. — Bender et al1 have collected a wealth of interesting data on the bilirubin-binding capacity of blood in premature infants. However, clarification of several issues related to the data set, as outlined below, would be helpful in understanding the stated conclusions. 1. Figure 1 of the article depicts the relationship between unbound bilirubin (UB) and total bilirubin (TSB) in 3 risk groups of premature infants: low (L), moderate (M) and high (H). This is a key figure underlying a principal finding of the article: that UB is higher in unstable than in stable neonates. Figure 1 is presented as representing the data listed in the accompanying Table 3 and refers to bilirubin measurements performed at 5 days of age and risk assessments at 24 hours of age. Table 3 shows that there were 31 (29%), 38 (36%), and 36 (34%) infants in the L, M, and H groups, respectively, for a total of 105 samples. However, examination of an enlarged PowerPoint slide of Fig 1 shows that, in disagreement with Table 3, there are many >105 data points on the plot. I counted at …

Bilirubin-binding Capacity in Premature Infants: In Reply

Thank you for raising many of the same methodological concerns that we wrestled with during the study design. We used “binding capacity” as an estimate of plasma bilirubin behavior for clinical, rather than experimental, comparisons. We sought to identify whether a reproducible, standardized, automated enzymatic measurement of free bilirubin under defined conditions would yield better predictions of risk than just total bilirubin. The use of “BBC estimate” should have been more …

Bilirubin-binding Capacity in Low Birthweight Premature Infants: In Reply

Thank you, Dr McDonagh, for your careful review of our study. The discrepancies between Fig 1 and Table 3 had textual description that was regrettably cut during our internal review cycle which, hopefully, I can reconcile now. 1. 1. Figure 1 has 138 data points, of which 52 (38%), 45 (33%), and 41 (30%) were in the high-risk (H), moderate-risk (M), and low-risk (L) categories at the first 24 hours. 2. Of 152 patients, 2 withdrew, 12 had no unbound bilirubin (UB), 9 had no Scatchard plot data, and 24 had poor primary site …

Ontogeny of Bilirubin-Binding Capacity and the Effect of Clinical Status in Premature Infants Born at Less Than 1300 Grams

Bilirubin is toxic to the brain and enters the brain in unbound form. Serum unconjugated, unbound bilirubin may be a good predictor of bilirubin encephalopathy. Unbound bilirubin levels may depend on the bilirubin-binding capacity of albumin, which has not been described for neonates of <28 weeks' gestation. The purpose of this work was to determine the ontogeny of bilirubin-binding capacity and the effect of clinical status in very preterm neonates. A total of 152 neonates (23-31 weeks' gestational age; 440-1300 g) were enrolled prospectively. At 5 days of age, total serum bilirubin and unbound bilirubin were measured with the unbound bilirubin-A1 analyzer (Arrows Co, Osaka, Japan) and albumin with the Bromocresol-purple method. Scatchard plots were used to estimate bilirubin-binding affinity and capacity. Clinical status for each infant was rated as high, moderate, or low risk by using a modified Score for Neonatal Acute Physiology model. Low risk was considered clinically stable. Unbound bilirubin has a significant, direct correlation to total bilirubin and is greater in unstable than in stable neonates. For the entire cohort, bilirubin-binding capacity had a direct relationship to gestational age. The bilirubin-binding capacities of infants in the low- and high-risk groups also had a direct relationship to gestational age. Bilirubin-binding capacity was greater in the low-risk group (20.8 +/- 4.6 mg/dL; 356 +/- 79 micromol/L) than in the moderate- (17.8 +/- 3.5 mg/dL; 304 +/- 60 micromol/L) or high- (17.3 +/- 3.4 mg/dL; 296 +/- 58 micromol/L) risk groups. Bilirubin-binding affinity did not differ by clinical risk status or gestational age. In very preterm, very low birth weight infants, bilirubin-binding capacity is directly proportional to gestational age. Bilirubin-binding capacity is lower and unbound bilirubin higher in unstable than in stable neonates. These data may be useful in guiding the management of hyperbilirubinemia in very low birth weight infants.

Calculated In Vivo Free Bilirubin Levels in the Central Nervous System of Gunn Rat Pups

In vitro studies suggest a free bilirubin (B(F)) concentration in the range of 71-770 nmol/L can induce neurotoxicity. In vivo data regarding central nervous system (CNS) B(F) levels have not been determined. We calculated in vivo CNS B(F) levels in Gunn rat pups (15-19 d old; heterozygous nonjaundiced Gunn rats (J/j) and homozygous jaundiced Gunn rats (j/j); saline or sulfadimethoxine treated) based on 1) total brain bilirubin (TBB) content, 2) brain albumin level, 3) CNS bilirubin binding capacity attributable to brain albumin determined using an ultrafiltration technique, and 4) published Gunn rat albumin-bilirubin binding constants (k). Gunn rat brain bilirubin binding capacity was approximately 22 x 10(-3) micromol/g, of which two thirds was accounted for by brain albumin. Using a Gunn rat pup in vivo, k of 9.2 L/micromol, calculated CNS B(F) levels ranged from 72 to 112 nmol/L [95% confidence interval (CI)] in saline and from 59 to 156 nmol/L (95% CI) in sulfadimethoxine-treated J/j pups. These animals demonstrated no neurobehavioral abnormalities and normal cerebellar weight. Calculated CNS B(F) levels were severalfold higher (p < 0.001) in saline (95% CI: 556-1110 nmol/L) and sulfadimethoxine-treated (95% CI: 3461-8985 nmol/L) j/j pups; the former evidenced reduced cerebellar weight; the latter both reduced cerebellar weight and acute neurobehavioral abnormalities. We conclude that calculated CNS B(F) values in j/j pups are substantially higher than those in J/j animals. Given the absence of CNS abnormalities in J/j pups, the presence of such in j/j animals, and the CNS B(F) levels in these groups, we speculate that the CNS B(F) neurotoxicity threshold in vivo is subsumed within the range (71-770 nmol/L) reported in vitro.

Binding of bilirubin with albumin-coupled liposomes: implications in the treatment of jaundice

In the present study, we demonstrated the suitability of liposomes as a method of removing plasma bilirubin in hyperbilirubinemic rats. The liposomes have innate tendency to bind with bilirubin through hydrophobic interaction. Among different types of liposomes, the positively charged liposomes were found to have maximum affinity to free bilirubin. However, the entrapment or coupling of serum albumin on the surface of egg phosphatidylcholine liposomes can render a several-fold increase in their bilirubin binding capacity. The proteoliposomes were able to preferentially bind with bilirubin even in the presence of erythrocytes. Interestingly, these liposomes were found to displace bilirubin bound on the surface of erythrocytes as well. The results of the present study further demonstrate that albumin-bearing liposomes were equally effective in removing plasma bilirubin in experimental jaundiced animals. These observations indicate that liposome-mediated selective homing of excess plasma bilirubin to the liver cells (cf. hepatocytes) may help in the development of safer strategy for the treatment of hyperbilirubinemic conditions in the model animals.

Preventing kernicterus: Almost there

Preventing kernicterus: Almost there

Decreased Bilirubin-Binding Capacity in Uremic Serum Caused by an Accumulation of Furan Dicarboxylic Acid

In chronic renal failure, substances that are effectively excreted in healthy subjects accumulate in serum. These substances, uremic toxins, include a variety of organic acids. It has been reported that a decrease in the bilirubin (BR) binding capacity occurs in the serum of renal failure patients. 3-Carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) has a high affinity for human serum albumin (HSA) and is a potent inhibitor of the serum protein binding of many drugs. We recently reported that CMPF and BR share the binding site for dicarboxylate molecules on the HSA molecule [Pharm Res 1999;16:916–923]. In this study, in order to confirm whether CMPF is involved in the decrease of BR serum binding capacity in chronic renal failure patients, the total concentrations of uremic toxins, CMPF, and indoxyl sulfate (IS) and the free BR concentration in serum from healthy volunteers and renal failure patients were determined. Both total CMPF and IS concentrations correlate with the free BR concentration. However, results from the peroxidase method reveal that IS cannot displace BR under the physiological condition [IS]/[HSA] <1. We, therefore, conclude that CMPF is one of the substances which contribute to the decreased binding capacity of BR in uremic serum.