Albumin-bound Bilirubin Research Articles

The fluorescence signal of albumin-bound bilirubin to monitor drug-induced bilirubin displacement from human serum albumin

Bilirubin (BR) binding to human serum albumin (HSA) may be affected by drugs, which may cause hyperbilirubinemia. A new method is proposed to investigate BR displacement from HSA using the characteristic fluorescence signal of albumin-bound BR at 533 nm upon 460 nm excitation. Any decrease in the fluorescence intensity at 533 nm upon drug addiction is direct evidence of BR displacement. Drug-induced BR displacement was tested using the well-known drug markers, warfarin (WFN) for Site I, ketoprofen (KTN) for Site II and hemin (HMN) for Site III of HSA. The maximum quenching (93%) and the highest Ka value (1.13 × 105 M1), obtained with HMN, suggested a common binding site of BR and HMN. Relatively lower BR displacement by WFN and KTN and lowered Ka values can be attributed to allosteric phenomena. These results showed the importance of the albumin-bound BR fluorescence signal in testing BR displacement from albumin.

The Effects of Clofibrate on Neonatal Jaundice: A Systematic Review.

Background:Neonatal jaundice is a prevalent disease that causes many complications, including kernicterus and even death. Previous studies have shown that clofibrate as an aryloxy isobutyric acid derivate can be effectively applied for the treatment of neonatal jaundice. Thus, this review was carried out to investigate the effects and mechanism of action of clofibrate on neonatal jaundice.Methods:The keywords such as “Clofibrate” in combination with “Neonatal jaundice” or “Neonatal hyperbilirubinemia” or “Newborn Jaundice” were used to search for relevant publications indexed in the Institute for Scientific Information (ISI), Scopus, PubMed, and Google Scholar databases. Finally, after reviewing the studies, 24 papers were included in this study.Results:Results showed that the processes of albumin-bound bilirubin transfer to the hepatocytes, hepatic uptake, and storage via ligandin, hepatic conjugation via uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), conjugation into the bile via MRP2 represent the main action mechanism of clofibrate that turns it into the bilirubin conjugates and expels it from the bile. Besides, clofibrate has been shown to reduce the level of Total Serum Bilirubin (TSB) in infants even at a dosage of 25 mg/kg without leaving side effects.Conclusions:The results of this review revealed that clofibrate effectively reduces TSB in short-term usage and can even have a promising effect at the dosage of 25 mg/kg in full-term infants. Most studies have shown this property over a short period in term infants, and there is no evidence about long-term usage in this regard.

Enhancement of the adsorption of bilirubin on activated carbon via modification

A commercial activated carbon that is based on coconut shell was modified to increase its mesopore size for the adsorption of albumin-bound bilirubin (AB bilirubin) and bilirubin. The characterization of activated carbon that was modified via liquid nitrogen adsorption indicated that its micropores were reduced in size and its mesopores enlarged. According to the adsorption kinetics results, its adsorption capacities for both AB bilirubin and bilirubin are substantially increased due to its enlarged mesopores. Along with the increased adsorption capacity, its adsorption rate to bilirubin is also substantially increased. The adsorption capacitiesfor bilirubin and AB bilirubin depend linearly on the cumulative mesopore volume in the size ranges of 2.4–4.1 ​nm and 8.0–30.0 ​nm, respectively. Additionally, bilirubin presents a faster adsorption rate than AB bilirubin.

Influence of the acid groups on activated carbon on the adsorption of bilirubin

In order to study the influence of the acid groups on activated carbon to the adsorption of bilirubin, a commercial activated carbon was modified and a series of activated carbons with varying amount of acid groups, along with their surface texture changed was obtained. Then the effect of acid groups and mesopores on the surface of modified activated carbons on adsorption of albumin-bound bilirubin and free bilirubin were investigated. Results indicated that the adsorption capacities of albumin-bound bilirubin were improved by the increasing acid groups introduced on the surface of modified activated carbons, in spite of the significantly reduced mesopores. The adsorption capacities of free bilirubin rise both with the elevated acid groups and mesopores.

High efficiency 3D nanofiber sponge for bilirubin removal used in hemoperfusion.

The accumulation of bilirubin in the body could cause nervous system diseases and even endanger life in severe cases for people with liver damage or metabolic obstruction. Hemoperfusion has been considered as one of the most efficient treatments to remove extra bilirubin. Although, the current bilirubin adsorbents could adsorb the free bilirubin effectively, the albumin-bound bilirubin in plasma is hard to remove. Here, we develop a 3D nanofiber sponge fabricated by combination of electrospinning and improved gas-foaming techniques. The amino groups and BSA molecules were immobilized on the fiber surface as the affinity groups to adsorb bilirubin. The 3D nanofiber sponges have layered structure and significantly higher porosity than two-dimensional nanofiber membranes. The special 3D structure renders the sponge fully contact with the adsorbed liquid and reduces the diffusion distance of the adsorbate, thus increases the sponge's adsorption rate. The BSA immobilized nanofiber sponge showed large adsorption capacity in both aqueous solution (maximum adsorption capacity was 36.8237 mg/g) and plasma (maximum adsorption capacity was 25.2908 mg/g), rapid adsorption rate (achieved adsorption equilibrium in 60 min) and well blood compatibility.

Albumin dialysis in artificial liver support systems: open-loop or closed-loop dialysis mode?

In artificial liver support systems, the open-loop albumin dialysis mode (OLM) is usually used to remove protein-bound toxins from the blood of patients with liver failure. However, there is still interest in the closed-loop albumin dialysis mode (CLM) because this mode may enable not only the regeneration and reuse of albumin but also the miniaturization of artificial liver systems. In this article, we compared the two modes under a fixed amount of albumin in dialysate experimentally and theoretically. The results show that according to the detoxification efficiency in the 3 hour dialysis for removing albumin-bound bilirubin, CLM is better than OLM. The usage efficiency of albumin in CLM is also higher. Moreover, the advantage of CLM is more significant when the concentration of bilirubin in blood is lower. Under a given amount of albumin in dialysate, if the concentration of bilirubin in blood is high, one may further increase the performance of CLM by means of increasing the flow rate of the albumin dialysate or using the highly concentrated albumin dialysate.

A New Method to Increase the Adsorption of Protein‐Bound Toxins in Artificial Liver Support Systems

In this work, a new method, called the preconcentration method (PCM), is proposed to increase the adsorption of protein-bound toxins onto adsorbents in artificial liver support systems. In the PCM, a concentrator is installed before the inlet of the adsorbent cartridge. This method is validated in an experiment using activated carbon to remove albumin-bound bilirubin, and the mechanism of the increase in adsorption is theoretically explained with breakthrough curve and equilibrium adsorption analyses. Our results show that when this PCM is used, the mass transfer rate of bilirubin from solution to activated carbon is enhanced, the adsorbed bilirubin amount per unit mass of activated carbon is greatly increased, and more albumin-bound bilirubin molecules are quickly removed from the albumin solution. When the concentration ratio (the ratio of the inlet flow rate to the outflow rate of the concentrator) is 2.59, the adsorption efficiency of activated carbon at 120 min is increased by approximately 36%. Only approximately 60 min is required for the bilirubin concentration to decrease from 19.3 to 13.0 mg/dL; however, without the PCM, nearly 180 min is needed. In addition, by adjusting the concentration ratio, the adsorption of albumin-bound bilirubin onto activated carbon can be further increased.

Serum bilirubin may serve as a marker for increased heme oxygenase activity and inducibility in tissues – A rationale for the versatile health protection associated with elevated plasma bilirubin

Unconjugated bilirubin functions intracellularly as a potent inhibitor of NADPH oxidase complexes, and albumin-bound bilirubin contributes significantly to the oxidant scavenging activity of plasma. So it is not surprising that serum levels of bilirubin have been found to correlate inversely with risk for vascular diseases and a host of other disorders. Nonetheless, recent Mendelian randomization analyses reveal that individuals who carry low expression alleles of the hepatic bilirubin conjugating enzyme UGT1A1, and hence have somewhat elevated levels of plasma bilirubin throughout life, are not at decreased risk for vascular disorders. This likely reflects the fact that, in most people, plasma levels of unconjugated, unbound bilirubin--the fraction of bilirubin capable of fluxing back into cells--are so low (near 1 nM) that they can exert only a trivial antioxidant influence on cells. In light of these findings, it is reasonable to propose that the inverse correlation of plasma bilirubin and disease risks noted in many studies often reflect the fact that elevated plasma bilirubin can serve as a marker for an increased propensity to generate bilirubin within cells. Consistent with this view, high expression alleles of the major enzymatic source of bilirubin, heme oxygenase-1 (HO-1), do associate with decreased vascular risk in the majority of studies that have addressed this issue, and increased plasma bilirubin has been reported in carriers of these alleles. Hence, the consistent reduction in vascular risk noted in people with Gilbert syndrome (traditionally defined as having serum bilirubin in excess of 20 μM) is likely attributable to an increased rate of bilirubin generation within tissues, rather than to the decreased hepatic UGT1A1 activity that characterizes this syndrome. However, there is good reason to suspect that, at some sufficiently high plasma bilirubin level--as in individuals with very intense Gilbert syndrome or in Gunn rats lacking UGT1A1 activity--the plasma bilirubin pool does indeed provide some antioxidant protection to cells. Strategies for boosting bilirubin production within cells via HO-1 induction, or for mimicking bilirubin's antioxidant activity with cyanobacterial phycobilins, may have important potential for health promotion.

Measuring Free Bilirubin: The Clinical Perspective

In 1959, Gerald Odell first proposed that albumin and tissue compete for binding bilirubin and that this competition is mediated by a very low concentration of free bilirubin (Bf)2 (bilirubin not bound to plasma proteins). He argued that displacement of albumin-bound bilirubin by binding competitors could explain the reported high incidence of kernicterus in premature infants receiving sulfonamide prophylaxis (1). This sentinel publication led to a flurry of proposed binding tests, most of which were nonspecific competitive-binding assays, until 1974, when Jacobsen and Wennberg presented a method for measuring Bf in infant plasma that was based on the observation that only unbound bilirubin serves as a substrate for enzymatic oxidation by horseradish peroxidase (2). Several studies from Japan, where the peroxidase assay had been adapted to a semiautomated instrument, identified a clear relationship of Bf with acute neurotoxicity in premature infants (3) and with acute auditory impairment in term infants (4). Enthusiasm was tempered in the US by a perceived difficulty in performing the assay and by confusion regarding the poor correlation between Bf and outcome in premature babies who died with kernicterus at very low total bilirubin (Bt) concentrations (5). The reported high incidence of death of infants with kernicterus was isolated to specific care centers: Very high Bf/Bt ratios in affected infants indicated very poor binding, and the phenomenon of lethal kernicterus at low Bt concentrations largely disappeared after cessation of benzyl alcohol use as a preservative in multiple-dose vials or solutions for parenteral therapy (6). Kernicterus is still occasionally observed at low Bt concentrations in babies with sepsis or …

Relationship between kinetics of albumin-bound bilirubin and water-soluble urea in extracorporeal blood purification

The purpose of the study was to examine the relationship between urea and conjugated bilirubin kinetics during extracorporeal liver support (ELS) therapy and to determine the dose of therapy for urea and conjugated bilirubin as markers for water-soluble and protein-bound solutes, respectively. Kinetics of urea and bilirubin were described by standard two-compartment models with central clearance, constant intercompartment clearance, constant generation rate and constant volume. While the concentration of urea was assumed as equilibrated between compartments at the beginning of ELS therapy, the concentration of conjugated bilirubin between compartments was assumed to follow the partition of albumin between plasma and interstitial spaces. Treatment dose was calculated as removed solute mass and fractional solute removal. Seven patients were studied during 15 treatments lasting at least 6 h. Bilirubin distribution volume of 14.8 ± 5.4 L was not different from urea extracellular water volume of 15.0 ± 2.8 L. The correspondence between models was used to predict the mass of bilirubin removed based on extracellular volume obtained from urea kinetics, average data from bilirubin kinetics, as well as selected treatment and patient information. The prediction of bilirubin mass removed based on this reduced information was not different from the mass of solute removed based on complete bilirubin kinetic analysis. The correspondence between kinetics of urea and conjugated bilirubin can be used to identify the bilirubin distribution volume from urea kinetic analysis. This information is then useful to estimate and predict the solute removal of conjugated bilirubin in ELS.

Limited Role for the Bilirubin-Biliverdin Redox Amplification Cycle in the Cellular Antioxidant Protection by Biliverdin Reductase

In mammalian cells, heme is degraded by heme oxygenase to biliverdin, which is then reduced to bilirubin by biliverdin reductase (BVR). Both bile pigments have reducing properties, and bilirubin is now generally considered to be a potent antioxidant, yet it remains unclear how it protects cells against oxidative damage. A presently popular explanation for the antioxidant function of bilirubin is a redox cycle in which bilirubin is oxidized to biliverdin and then recycled by BVR. Here, we reexamined this putative BVR-mediated redox cycle. We observed that lipid peroxidation-mediated oxidation of bilirubin in chloroform, a model of cell membrane-bound bilirubin, did not yield biliverdin, a prerequisite for the putative redox cycle. Similarly, H(2)O(2) did not oxidize albumin-bound bilirubin to biliverdin, and in vitro oxidation of albumin or ligandin-bound bilirubin by peroxyl radicals gave modest yields of biliverdin. In addition, decreasing cellular BVR protein and activity in HeLa cells using RNA interference did not alter H(2)O(2)-mediated cell death, just as BVR overexpression failed to enhance protection of these cells against H(2)O(2)-mediated damage, irrespective of whether bilirubin or biliverdin were added to the cells as substrate for the putative redox cycle. Similarly, transformation of human BVR into hmx1 (heme oxygenase) mutant yeast did not provide protection against H(2)O(2) toxicity above that seen in hmx1 mutant yeast expressing human heme oxygenase-1. Together, these results argue against the BVR-mediated redox cycle playing a general or important role as cellular antioxidant defense mechanism.

Capillary electrophoresis for the detection of bilirubin in rat serum

The rat is used often to assess the toxicity of new chemical entities in preclinical drug development. Bilirubin concentration in rat serum is routinely determined by colorimetric methods, but false positive results due to hemolyzed serum or direct interferences by test compounds may occur. Capillary electrophoresis (CE) is an automated method that requires small sample volume and facilitates the direct detection of bilirubin. The purpose of this study was to evaluate a CE method for detecting bilirubin and albumin-bound bilirubin in rat serum and to measure potential interference by hemolysis and specific test compounds. Serum samples from male Sprague Dawley rats (n=20) were used in the study. Results obtained on a Beckman P/ACE MDQ CE instrument equipped with a UV-detector were compared with those obtained using a colorimetric method on a Hitachi 912 analyzer. Bilirubin standards were used to evaluate the detection and stability of bilirubin in rat serum, and vials with ultrafiltration membranes were used to separate albumin-bound bilirubin. Intraday and interday coefficients of variation (CV), linearity, and the effects of added hemoglobin and a test compound on CE results were determined. The CE method was capable of detecting bilirubin and albumin-bound bilirubin in rat serum samples with reproducible results and good accuracy. CVs were <3% and linearity of the CE assay was high (R(2)=0.9951). Abnormally high bilirubin peaks due to the presence of hemoglobin or the test compound were easily distinguished by means of CE. CE is a good alternative to the colorimetric methods currently used for the determination of bilirubin in rat serum.

Unbound Bilirubin: A Better Predictor of Kernicterus?

The peroxidase method described in this paper measures non–protein-bound bilirubin by oxidation of the free fraction. The oxidation rate is slow compared with rapid dissociation of albumin-bound bilirubin. Thus, the unbound bilirubin can be estimated from the initial rate of decline in total bilirubin concentration. In the 1970s, severe neonatal hyperbilirubinemia and kernicterus were major clinical problems. Kernicterus could usually be prevented by administering an exchange transfusion to infants whose serum unconjugated bilirubin concentration reached 341 μmol/L (20 mg/dL), but the diagnostic specificity of 341 μmol/L (20 mg/dL) was quite poor. Premature infants sometimes developed encephalopathy at very low bilirubin concentrations, especially when receiving sulfonamides. While engaged in a fellowship in neonatal medicine, I was stimulated by Gerald Odell’s 1959 paper, in which he used pharmacological arguments to explain how these drugs produced kernicterus by …

CD163-Mediated Hemoglobin-Heme Uptake Activates Macrophage HO-1, Providing an Antiinflammatory Function

See related article, pages 943–950 A study by Schaer et al,1 which appears in this issue of Circulation Research , provides evidence suggesting that CD163 acts as a hemoglobin (Hb) transporter and, via activation of macrophages, heme oxygenase-1 (HO-1) provides antiinflammatory activity, presumably by an increase in ferritin. Induction of HO-1 was observed to occur when the cultured macrophages, endothelial cells or tubular cells were exposed to heme or hemoglobin (for a review see Abraham and Kappas2). Heme and denatured hemoglobin toxicity play an important role in a broad spectrum of pathological circumstances such as myocardial ischemia, hypertension, cardiomyopathy, reperfusion, organ transplantation, pulmonary disorders, and inflammation among others.3 In the cell free system, hemoglobin is bound to haptoglobin before clearance by the macrophage hemoglobin scavenger receptor CD163. Schaer et al1 demonstrated that CD163-hemoglobin transport is regarded as a critical step in the hemoglobin clearance pathway in the macrophage, especially under conditions of extreme hemoglobin release resulting from hemolysis. Binding hemoglobin-haptoglobin to CD163 cells also elicits IL-10 secretion, which contributes to the induction of HO-1.4 Thus, CD163 has a functional role as an antioxidant by enhancing HO-1, the major cytoprotective response. This links heme transport directly to a known receptor with well characterized endocytic properties and signaling functions.5 Heme-hemopexin is also taken up by LRP/CD91 and, as such, may inhibit inflammatory functions in phagocytic and antiinflammatory macrophages; however, a link to functional HO-1 was not described. Schaer et al1 demonstrated that induction of HO-1 by Hb is functionally linked to CD163 and, using gene array analysis, were able to show that Hb elicits a noninflammatory transcriptional response in macrophages, presumably by the coordinated increase in ferritin synthesis following the activation of HO-1. This response was mediated by CD163 and did not involve the …

Albumin-Bound Bilirubin Interacts with Nitric Oxide by a Redox Mechanism

Bilirubin, the final product of heme catabolism, plays a crucial role in the cellular defense against oxidative and nitrosative stress. This study investigated the interaction of albumin-bound bilirubin, the circulating form of the bile pigment, with nitric oxide (NO), a gaseous modulator involved in many physiological functions but able to induce cytotoxicity and cell death if produced in excess. A short-lived endogenous S-nitrosothiol such as S-nitroso-cysteine was used as NO donor. In PBS without chelators, bilirubin was bound to human serum albumin with an apparent affinity of 1.6 +/- 0.2 microM (n = 4). Furthermore, albumin (2-20 microM) dose-dependently increased the half-life of BR (10 microM) exposed to S-nitroso-cysteine (100 microM) of 2.4 +/- 0.4 times (n = 4). Albumin-bound bilirubin was almost completely oxidized by S-nitroso-cysteine-derived NO, and biliverdin was the major product formed; this reaction seemed to be rather specific for albumin-bound bilirubin because when free bilirubin was reacted with S-nitroso-cysteine the formation of biliverdin was significantly lower. Uric acid and reduced glutathione, two well-known plasma antioxidants, at physiological concentrations protected albumin-bound bilirubin from NO-mediated oxidation. Taken together, these data suggest that albumin-bound bilirubin maintains its ability to interact with NO also in the bloodstream counteracting extracellular nitrosative reactions.

Characterization of the oxidation products of BO-653 formed during peroxyl radical-mediated oxidation of human plasma

4,6-Di- tert-butyl-2,3-dihydro-2,2-dipentyl-5-benzofuranol (BO-653) is a novel antioxidant synthesized by theoretical findings and considerations. Here we report on the aqueous peroxyl radical-induced oxidation of human plasma in the presence of BO-653. When BO-653 was given to healthy human subjects at 400 mg twice daily for 28 days, lipids in the resulting plasma were protected from oxidation compared with lipids present in plasma from subjects receiving placebo. Similarly, BO-653 added in vitro at 50 μM inhibited the peroxyl radical-induced accumulation of cholesteryl ester hydroperoxides that occurred in the presence of α-tocopherol, although BO-653 did not decrease the rate of consumption of ascorbate, albumin-bound bilirubin, and uric acid. The antioxidant action of in vivo and in vitro added BO-653 was associated with the formation of two major reaction products of BO-653, the structures of which were elucidated by mass spectrometry and nuclear magnetic resonance analyses. The products were identified as stereoisomers of dioxybis(4,6-di- tert.-butyl-2,3,5,7 a-tetrahydro-2,2-dipentylbenzofuran-5-one). These dialkylperoxides of BO-653 might be useful markers to assess the antioxidant function of BO-653 in biological systems in vivo.

Nonregenerative stimulation of hepatocyte proliferation in the rat: variable effects in relation to spontaneous liver growth; a possible link with metabolic induction.

Three procedures were used to stimulate hepatocyte proliferation in the rat without reducing liver mass, resulting in a supplementary growth which differs from the regenerative growth observed after loss of liver mass by hepatectomy or toxic necrosis. They were: (a) the ingestion of cyproterone, a cytochrome P450 inducing drug (b) the injection of an irritant which provokes glycogenesis and synthesis of acute-phase proteins (c) the injection of albumin-bound bilirubin leading to elimination of glucuronated bilirubin in bile. This ensuing supplementary growth was studied in the rat under several conditions of hepatic proliferation: 1. In normal adult rats, in which hepatocyte proliferation is very low, the effect on proliferation was either weak or undetectable. 2. In suckling rats, with a rapid body and liver growth, all the stimulants provoked a synchronized wave of proliferation with a steep increase of the percentage of S-phase hepatocytes from 4.5% in controls to 15-30% in treated rats. This increase was followed by a compensatory period of low proliferation during which a treatment with a second stimulant was much less effective. 3. In 2/3 hepatectomized adult rats, the proliferation induced by cyproterone was higher than the spontaneous regenerative proliferation alone and additional to it during all of the regenerative process. The proliferation induced by acute inflammation was competitive with the synchronous spontaneous proliferation during the early period of synchronized proliferation following surgery, suggesting that both are similar acute responses. Differently, during the late period of lower and unsynchronized regenerative proliferation, the proliferation provoked by acute inflammation was additional to the spontaneous one. A stimulation of proliferation by injection of the albumin-bilirubin complex was observed during the late period after 2/3 hepatectomy. The highest level of stimulation occurred when the liver growth and the hepatocyte proliferation were already high. This suggests that these stimulants are not complete mitogenic stimuli and need cofactors which are present during the spontaneous growth or, alternatively, that the effect of stimulants is opposed by an inhibitory mechanism present in the adult rat.

On the modulation of photoinduced fluorescence enhancement and conformational stability of albumin-bound bilirubin:: Effect of ϵ-NH2 groups blocking and chloroform binding

Photoinduced fluorescence enhancement of bilirubin bound to primary binding site on human serum albumin (HSA) was completely ceased when ϵ-NH2 groups of its internal lysine residues were covalently blocked by acetylation or succinylation though the pigment bound to these derivatives in a folded conformation akin to that bound to HSA. These photoinduced fluorescence modulations cannot be ascribed to the binding of bilirubin to secondary low affinity sites as the CD spectrum of bilirubin bound to these derivatives showed complete inversion upon addition of chloroform which binds to subdomain IIA in HSA where high affinity bilirubin binding site is located. Presence of chloroform reconciled the photoinduced alterations in the CD spectrum observed in its absence, suggesting that chloroform stabilized the bound ligand against light but the fluorescence properties of bilirubin complexed with acetylated or succinylated derivatives remained unchanged. Guanidination of internal ϵ-NH2 groups in HSA by O-methylisourea did not alter the spectral properties of the bound ligand. These results suggest that salt linkage(s) existing between ϵ-NH2 groups of lysine residues in HSA and carboxyl groups of bilirubin, act(s) as a potential barrier during conformational rotation of the bound ligand assisted by photoactivation and their abolishment can alter its dynamics and stereoselectivity, a hitherto unnoticed implication of salt linkage(s) in BR–HSA complex.

Effects of bilirubin and its photoisomers on direct bilirubin measurement using bilirubin oxidase

We examined the reactivity of human serum albumin-bound bilirubin and its photoisomers as substrates for a direct bilirubin assay using bilirubin oxidase. The reduction of (EZ)-cyclobilirubin reached 100% 5 min after addition of the enzyme at any pH tested (3·5–7·4) in 0·1 mol/L phosphate buffer, whereas the reduction of (ZE)-bilirubin or (ZZ)-bilirubin reached 100% only below pH 4·5 or 5·5, respectively. (ZZ)-Bilirubin and its photoisomers did not react in citrate-lactate buffer at pH 3·7. The circular dichroism spectrum of (ZZ)-bilirubin in this buffer did not show a positive Cotton effect. These results indicate that a three-dimensional structure surrounding the reaction site of bilirubin is important for the reactivity with bilirubin oxidase.

Effects of bilirubin and its photoisomers on direct bilirubin measurement using bilirubin oxidase.

We examined the reactivity of human serum albumin-bound bilirubin and its photoisomers as substrates for a direct bilirubin assay using bilirubin oxidase. The reduction of (EZ)-cyclobilirubin reached 100% 5 min after addition of the enzyme at any pH tested (3.5-7.4) in 0.1 mol/L phosphate buffer, whereas the reduction of (ZE)-bilirubin or (ZZ)-bilirubin reached 100% only below pH 4.5 or 5.5, respectively. (ZZ)-Bilirubin and its photoisomers did not react in citrate-lactate buffer at pH 3.7. The circular dichroism spectrum of (ZZ)-bilirubin in this buffer did not show a positive Cotton effect. These results indicate that a three-dimensional structure surrounding the reaction site of bilirubin is important for the reactivity with bilirubin oxidase.