Gunn rats: a reproducible experimental model to compare the different methods of measurements of bilirubin serum concentration and to evaluate the risk of bilirubin encephalopathy

Three groups of Gunn rats were studied: group 1 was perfused with bilirubin solution alone, group 2 was perfused with bilirubin and albumin solutions simultaneously, group 3 was perfused with bilirubin solution for 30 min then bilirubin and albumin solutions for the following 10 min. Our results indicate that (1) Gunn rats are a reliable experimental model to study the risk of bilirubin encephalopathy, (2) unbound bilirubin can enter the brain when albumin binding capacity is reduced, (3) and bilirubin binding capacity of serum for unbound unconjugated serum bilirubin is a better criterion than total serum bilirubin and erythrocyte bilirubin to evaluate the risk of kernicterus. This model could also be used to study variations of permeability of the blood-brain-barrier and influences of drugs on bilirubin metabolism.     

The Electrophoretic Mobility of Chylomicrons in a Cask of Essential Hyperlipemia

The absorptivity of alkaline solutions of highly purified bilirubin has been determined. The concentration of bilirubin in alkaline standard solutions of commercially available bilirubin may then be determined by direct spectrophotometry.

The absorptivity of bilirubin at four different pH levels in normal sera is given.     

Interaction of beta-lactam antibiotics on bilirubin-albumin complex: comparison by three methods, total bilirubin, unbound bilirubin and erythrocyte-bound bilirubin

The effects of 'third-generation' cephalosporins and penicillin analogues on the concentrations of total unconjugated bilirubin, unbound bilirubin and erythrocyte-bound bilirubin were determined in blood samples. This study was performed, in vitro, at two bilirubin/albumin molar ratios and at various concentrations of antibiotics. The most effective displacers, considering the three methods, were antibiotics tightly bound to albumin: ceftriaxone and cefotetan. Cefoperazone, which is bound to albumin as tightly as these two antibiotics, caused no significant increase in unbound bilirubin but should be considered as a displacer drug on the basis of the variations of erythrocyte-bound bilirubin and total bilirubin. We suggest that drug interaction on bilirubin-albumin binding be investigated by several methods.     

Assessment of bilirubin toxicity to erythrocytes. Implication in neonatal jaundice management

BACKGROUND: Neonatal hyperbilirubinaemia remains one of the most common clinical conditions requiring therapeutic intervention. Nevertheless, reliable indicators of bilirubin toxicity are still missing. This prompted us to investigate (a) the progression of cytotoxic events produced by increasing concentrations of bilirubin; (b) the relevance of the membrane lipid package on bilirubin binding to erythrocytes; and (c) the reliability of chloroform extraction compared with albumin extraction to evaluate erythrocyte-bound bilirubin and cytotoxicity. MATERIALS AND METHODS: Morphological alterations, free bilirubin, erythrocyte-bound bilirubin (albumin- and chloroform-extractable), haemolysis and membrane-released lipids, were determined in human erythrocytes at 4 degrees C or 37 degrees C, after 4 h incubation at pH 7.4, with increasing molar ratios of bilirubin to albumin (0.5-5). The reversibility of cytotoxicity by albumin washing was assessed by morphological analysis. RESULTS: Decreased free bilirubin, lower erythrocyte-bound bilirubin concentration by albumin extraction (superficial/non-aggregated bilirubin) and higher values by chloroform extraction (deep/aggregated bilirubin) were observed for 37 degrees C vs. 4 degrees C, at molar ratios > 1. Echinocytosis increased with bilirubin concentration and temperature and was not fully reversed by albumin washing. Haemolysis was already significant at a molar ratio of 1, and was enhanced by temperature at molar ratios 3 and 5 (P < 0.01). The loss of membrane lipids was remarkable at molar ratios > or = 0.5, both at 4 degrees C and 37 degrees C (P < 0.01), although correlation with bilirubin concentration was only significant at 37 degrees C (r = 0.971; P < 0.01). CONCLUSIONS: These results suggest that increased lipid fluidity and high bilirubin concentrations promote membrane bilirubin translocation and toxicity. They also show that albumin is not able to displace the bilirubin located deeply or aggregated within the membrane, which in turn is removed by chloroform. Accordingly, chloroform-extractable rather than albumin-extractable bilirubin is a more accurate parameter to assess erythrocyte-bound bilirubin during severe hyperbilirubinaemia.     

Spectral characteristics of bilirubin-bovine albumin complexes

Spectral characteristics of bilirubin-bovine albumin complexes have been examined. In the presence of excess albumin, the absorption spectrum of the complex did not change from pH 11.5–8.5. As the pH decreased from 8.5 to 5.5, there was a concomitant shift in the absorption maximum from 472 nm to 454 nm, and the spectra intersected at an isobestic point. This transition included the physiologically important pH range and had a pK = 6.6. Between pH 4.5 and pH 3.4 a complex with maximum absorbance at 421 nm was formed and appeared to be dependent on the N-F isomer transformation of albumin.At pH 5.1, there was spectral evidence that 1 mole of albumin binds only 1 mole bilirubin, since difference spectra of bilirubin-albumin solutions containing excess bilirubin were similar to the spectrum of unbound bilirubin. At pH 8.5, 1 mole albumin would appear to bind 1 mole bilirubin with an absorption maximum at 472 nm, although difference spectra indicated that albumin forms additional complexes with an absorption maximum at 424 nm.     

Hepatic bilirubin uptake in the isolated perfused rat liver is not facilitated by albumin binding.

Bilirubin uptake by the liver is a rapid process of high specificity that has kinetic characteristics which suggest carrier-mediation. In the circulation, bilirubin is readily bound to albumin, from which it is extracted by the liver. Although several studies suggested that it is the small, unbound fraction of bilirubin which interacts with hepatocytes and is removed from the circulation, recent experiments have been interpreted as suggesting that binding to albumin facilitates ligand uptake. A liver cell surface receptor for albumin has been postulated. The present study was designed to examine directly whether albumin facilitates the hepatic uptake of bilirubin and whether uptake of bilirubin depends on binding to albumin. Rat liver was perfused with a protein-free fluorocarbon medium, and single-pass uptake of 1, 10, or 200 nmol of [3H]bilirubin was determined after injection as an equimolar complex with 125I-albumin, with 125I-ligandin, or free with only a [14C]sucrose reference. Uptake of 10 nmol of [3H]bilirubin was 67.5 +/- 3.7% of the dose when injected with 125I-albumin, 67.4 +/- 6.5% when injected with 125I-ligandin, and 74.9 +/- 2.4% when injected with [14C]sucrose (P greater than 0.1). At 200 nmol, uptake fell to 46.4 +/- 3.1% (125I-albumin) and 63.3 +/- 3.4% [( 14C]sucrose) of injected [3H]bilirubin (P less than 0.01), which suggests saturation of the uptake mechanism. When influx was quantitated by the model of Goresky, similar results were obtained. When [3H]bilirubin was injected simultaneously with equimolar 125I-albumin and a [14C]sucrose reference, there was no delay in 125I-albumin transit as compared with that of [14C]sucrose. This suggested that the off-rate of albumin from a putative hepatocyte receptor would have to be very rapid, which is unusual for high affinity receptor-ligand interaction. There was no evidence for facilitation of bilirubin uptake by binding to albumin or for interaction of albumin with a liver cell surface receptor. These results suggest that the hepatic bilirubin uptake mechanism is one of high affinity which can extract bilirubin from circulating carriers such as albumin, ligandin, or fluorocarbon.     

Studies with I131-Labelled Fat1

The effect of free fatty acids (FFA), bile acids, and hematin on bilirubin binding by erythrocytes was investigated by adding increasing concentrations of these substances to incubation mixtures of bilirubin, albumin, and erythrocytes. It was found that both cholic acid and taurocholic acid increased the cellular binding of bilirubin. With bilirubin/albumin ratios below unity this effect was greatly potentiated by reducing the pH from 7.4 to 7.0–6.8. Hematin also increased the cellular binding of bilirubin; this effect seemed not to be potentiated by reduction in pH. With bilirubin/albumin ratios below unity, oleate was also found to increase cellular binding of bilirubin when the molar FFA/albumin ratio exceeded 6. With bilirubin/albumin ratios above unity, however, increased amounts of FFA reduced the cellular binding of bilirubin, especially at low pH. The findings and their possible clinical significance are discussed.     

A Standardized Direct Method for Total Cholesterol Determination in Serum with a Combined Reagent

Abstract

A clonal strain of rat hepatoma cells (MH₁C₁) known to take up and conjugate bilirubin was incubated in media containing bilirubin and albumin in different molar ratios at a constant bilirubin concentration. The highest rate of bilirubin conjugation was found at a molar ratio of 1/1. Excess bilirubin markedly reduced both the conjugation of bilirubin and the incorporation of [¹⁴C]alanine, indicating a toxic effect on the cells. Excess albumin also depressed the formation of bilirubin conjugates, but incorporation of [¹⁴C]alanine proceeded at a normal rate, indicating that the reduced conjugation is probably due to stronger binding of bilirubin to albumin and reduced cellular uptake of bilirubin. Bilirubin bound to albumin at a molar ratio of 1/1 had no toxic effects on the cells as judged by cell morphology and conjugation capacity, even at a total bilirubin concentration as high as 680 μmol/l.     

Bilirubin diglucuronide as the main source for in vitro formation of delta bilirubin

To clarify which of the bilirubin moieties is responsible for the formation of bilirubin bonded to albumin (delta bilirubin) in icteric serum, the in vitro formation of delta bilirubin from bile acid-free bilirubin glucuronides and unconjugated bilirubin was examined by high-performance liquid chromatography. Bovine serum albumin (150 mumol/liter) was mixed with equimolar bilirubin diglucuronide (BDG), bilirubin monoglucuronide (BMG), or unconjugated bilirubin (UCB) and incubated in the dark at 37 degrees C under argon gas saturation. Although no delta bilirubin was formed immediately, formation eventually occurred and increased with time. A similar amount of delta bilirubin was formed when human serum albumin was used instead of bovine serum albumin. Of the three types of bilirubin, BDG was found to be the greatest source of delta bilirubin, whereas UCB produced the least. On the other hand, photoirradiation of a mixture of bovine serum albumin and UCB at a molar ratio of 1:1 resulted 6 hr later in the formation of three times as much delta bilirubin as in nonirradiated specimens. This photoinduced delta bilirubin formation increased further when the UCB/albumin molar ratio was increased to 2:1.     

Non-resolving jaundice: bilirubin covalently attached to serum albumin circulates with the same metabolic half-life as albumin

In hepatobiliary disease and biliary obstruction, bilirubin often becomes covalently bound to albumin circulating in serum, producing a nondissociable complex. To determine how long this complexed bilirubin remains in the circulation, we compared the metabolic clearance of bilirubin-albumin complexes with the clearances of free bilirubin and unmodified albumin. Radiolabeled bilirubin, albumin, and covalent bilirubin-albumin were injected into the circulation of Sprague-Dawley rats and serial samples of plasma were analyzed for the injected compounds. The half-life of bilirubin was 6.2 min. The half-life of bilirubin covalently bound to rat serum albumin was 1.9 to 2.1 days, identical to that of unmodified rat albumin. We conclude that bilirubin covalently attached to albumin is maintained in the circulation with the long half-life of albumin rather than the short half-life of bilirubin. Because albumin in humans has a half-life of 19 days, covalent attachment of bilirubin to human albumin could result in persistence of hyperbilirubinemia long after the resolution of disease.     

Microdetermination of unbound bilirubin in icteric newborn sera: An enzymatic method employing peroxidase and glucose oxidase

An enzymatic assay method for the microdetermination of unbound bilirubin in newborn icteric sera is described. Unbound bilirubin is oxidized to colorless compounds by peroxidase in the presence of hydrogen peroxide derived from glucose by the mediation of glucose oxidase. In this method, the bilirubin is not significantly degraded before the addition of peroxidase, in contrast to the method using hydrogen peroxide. The oxidation rate is determined by spectrophotometry and chloroform extraction is eliminated.The unbound bilirubin concentration can be determined fromthe initial oxidation velocity of total bilirubin. The Michaelis constant, $\text{K}{}_{\mathrm{M}}$ was approximately 20 μM. The coefficient of variation for icteric serum determination was 4.4–6.5%. The concentration of unbound bilirubin was reduced after five days of storage at ?20° C.The bilirubin-albumin binding affinity was studied with purified albumin and adult serum. The dissociation constants were 2 × 10^?8 M and 5 × 10^?9 M, respectively, at bilirubin/albumin molar ratios below 1.0.Clinically, serum samples from 75 icteric newborn infants were analysed, and the sera of premature infants were found to have remarkably high levels of unbound bilirubin compared to those of fullterm infants. The sera of a Rhesus immunization infant and an ABO incompatibility infant were remarkably higher than that of the nonhemolytic icteric sera. The unbound bilirubin concentration was also affected, in an in vitro study, by the addition of hemolysate.     

应用全自动生化分析仪测定缺血修饰白蛋白

目的建立白蛋白钴结合(ACB)试验检测缺血修饰白蛋白(IMA)。方法应用日立7600型全自动生化分析仪,采用ACB法测定IMA,并测定血清中不同白蛋白浓度,以观察白蛋白对其影响。结果ACB方法批内变异系数(CV)为5.0%,批间CV为1.6%。不同浓度的人血清白蛋白ACB法测定结果是线性的,回归方程为Y=0.029 1.57X,r=0.995,P=0.000。黄疸对测定无明显影响;溶血(5 g/dL)和脂血(0.4%)对测定有干扰。健康人群参考范围为79.6 U/mL。结论IMA是评价心肌缺血的有效诊断工具,采用全自动生化分析仪建立测定IMA的方法方便、简单,可以大批量测定。     

The albumin binding of unconjugated bilirubin in serum.

1. The limited bilirubin binding capacity of human serum albumin, and the fact that kernicterus can occur once the serum unconjugated bilirubin concentration exceeds this capacity, makes the assessment of non-albumin bound free bilirubin valuable in cases of severe neonatal hyperbilirubinemia. 2. Present methodology for this assessment utilizes Sephadex column chromatography, and is somewhat tedious and slow. 3. We have developed a procedure for assessing the albumin binding capacity of serum by titrating a sample of the serum with T-20 Dextran coated charcoal. 4. The method requires 2 ml of serum, takes 90 minutes to complete and is highly reproducible. 5. By this method, we can determine the reported secondary loose binding capacity of the albumin as well as the tight binding capacity which is determined by existing methods. 6. The tight binding capacity of a pool of normal adult human serum was found to be 20 mg/dl of serum. 7. This is in agreement with existing methods. The loose binding capacity was found to be an additional 10 mg/dl of serum. Added phenobarbital was found to lower the tight binding capacity, but not the secondary capacity.     

Enzymatic determination of bilirubin fractions in serum

Novel enzymatic methods using bilirubin oxidase from Myrothecium verrucaria are described for the determination of bilirubin fractions in serum. These fractions include an unconjugated form (Bu), a conjugated form (Bc), and a delta fraction of bilirubin that reacts with direct diazo reagent and is irreversibly bound to serum albumin (Bδ). The determination is based on the different reactivities of the enzyme to bilirubin fractions at different pH in the presence or absence of anionic detergent such as sodium dodecyl sulfate (SDS) or sodium cholate. Thus, in the absence of detergents, Bc and Bδ are oxidized at acidic pH, while Bc is oxidized at alkaline pH; Bu is not oxidized at either acidic or alkaline pH.

The first approach is based on measuring the decreased absorbance of bilirubin colour at 450 nm caused by the enzymatic oxidation. Total bilirubin is measured at pH 8.0 in the presence of SDS. Direct bilirubin is measured at pH 3.7 and Bc is measured at pH 10.0 in the absence of SDS, respectively.

The second approach is made by coupling the diazo reaction with the enzyme reaction. Total and direct bilirubin are determined by a conventional diazo method without prior treatment by the enzyme. Bδ is determined with a direct diazo method after the serum sample is treated at pH 10.0 by the enzyme to oxidize the Bc fraction. The precision and the accuracy of these methods were compared with a precision and the accuracy of these methods were compared with a diazo method, an HPLC method and a slice method, and good results were obtained.     

Origin of mammalian biliprotein and rearrangement of bilirubin glucuronides in vivo in the rat.

In hepatobiliary disease bilirubin becomes bound covalently to serum albumin, producing a nondissociable bile pigment-protein complex (biliprotein). To elucidate the mechanism of biliprotein formation we studied the bile pigment composition of blood from animals with experimental cholestasis and carried out comparative studies on the rate of biliprotein formation in vivo and in vitro during incubation of bilirubin glucuronides with albumin. Bile duct ligation in the rat and guinea pig led to rapid accumulation in the circulation of bilirubin, heterogeneous bilirubin esters of glucuronic acid, and a biliprotein that migrated along with albumin on high performance liquid chromatography. When the obstruction was removed, biliprotein remained longer in the circulation than did the other bile pigment species. Biliprotein and heterogeneous bilirubin esters of glucuronic acid were not formed in bile duct-ligated homozygous Gunn rats but they were formed when bilirubin glucuronides were incubated with Sprague-Dawley rat serum or human serum albumin at 37 degrees C in vitro. Bilirubin glucuronide rearrangement in vitro was accompanied by nonenzymic hydrolysis. We conclude that the formation of biliprotein in vivo is probably nonenzymic and suggest that mammalian biliprotein is formed by acyl migration of bilirubin from a bilirubin-glucuronic acid ester to a nucleophilic site on albumin.     

Bilirubin Conjugation in the Human Fetus

Quantitative determination of bilirubin (unconjugated, by chloroform extraction), bilirubin diglucuronide (by the isotope derivative method) and albumin was done in 26 specimens of amniotic fluid from cases suspected of hemolytic disease in utero.

A hypothetical scheme of fetal bilirubin metabolism is proposed, presuming unidirectional transplacental elimination of unconjugated bilirubin. On the basis of theoretical considerations of the mechanism of conjugation it is thought possible that bilirubin in the fetal liver is in chemical equilibrium with its diglucuronide. Both pigments are in equilibrium with the pigments in the amniotic fluid. Equilibration takes place through the fetal plasma. The ratio of concentrations of bilirubin and bilirubin diglucuronide in the amniotic fluid is determined by the following three factors: the relative affinities of the binding of the two pigments to albumin, the ratio of concentrations of UDPGA to UDP in the liver, and the standard free energy of conjugation. The slow glycogen synthesis in the fetal liver may be responsible for a high ratio of UDPGA to UDP and hence for a high proportion of conjugated bilirubin in amniotic fluid.

The experimental findings are in agreement with this hypothesis, offering an alternative to the generally accepted conception of low transferase activity and impaired hepatic excretion of conjugate before and shortly after birth.     

Chemical nature of a synthetic bilirubin conjugate and its reactivities in the total and direct reactions by the Jendrassik-Gróf method

Using liquid chromatography, nuclear magnetic resonance spectroscopy, and elemental analyses, we verified that a commercially available synthetic bilirubin conjugate is predominantly a ditaurobilirubin (DTB) disodium salt. The Jendrassik-Gróf total bilirubin (TBIL) method quantitatively measures unconjugated bilirubin (Bu) and the Bu-equivalent content in DTB, from which we infer that the azopigments of Bu and DTB have identical molar absorptivities, which do not change in the presence of either serum or serum albumin of human or bovine origin. However, based on the ratio of direct bilirubin (DBIL) to TBIL, the DBIL reaction in dilute HCI is incomplete (even up to 20 min), with lower yields in a matrix of bovine serum albumin than in human serum. By contrast, the DBIL reaction in acetate buffer (pH 4.75) is quantitative for DTB in human serum (or its albumin), but less so in bovine serum (or its albumin). DTB is water soluble, is relatively stable when lyophilized with human serum, and is determined with such high precision in the above-mentioned endpoint assays that it may be a suitable surrogate calibrator for conjugated bilirubin.     

The determination of bilirubin with a new enzymatic method (Dri-STAT bilirubin) using the Hitachi 704 selective analyzer

A new enzymatic method for the determination of bilirubin in serum and plasma by means of a Hitachi 704 selective analyzer was evaluated. This endpoint method (37 degrees C) including a sample blank showed very reliable results. The range of linearity was 0.3 to 437 mumol/l bilirubin. The within-run imprecision of three different bilirubin concentrations (n = 16) was 0.37, 0.44 and 0.76% (coefficient of variation). Between-assay imprecision (n = 15) was 0.51 to 1.76% (coefficient of variation) for five different control materials. Inaccuracy, determined with 5 control sera (assigned values: 19, 23.9, 90.7, 142.0 and 295.6 mumol/l bilirubin), was 0.14 to 4.27%. Recovery rates, determined in two spiked plasma samples, were 97.8% and 99.1%, and in six bilirubin standard solutions between 92 and 99%. The comparison with the routinely used 2.5-dichlorophenyl diazonium salt method as well as with the Jendrassik & Grof [1938) Biochem. Z. 297, 81-89) method as the reference yielded correlation coefficients of r = 0.997 and r = 0.998.     

The interactions between iophenoxic acid,iopanoic acid,bilirubin and human serum albumin as studied by fluorescence and sephadex gel filtration

Iophenoxic acid increases the fluorescence of bilirubin bound to human serum albumin at drug/albumin molar ratios lower than 1, while iopanoic acid decreases it. The fluorescence enhancement results probably from a change in the fluorescence efficiency due to an iophenoxic acid-induced conformational change in the albumin, which in turn causes displacement of bilirubin from the protein. Iophenoxic acid does not affect the high-affinity bilirubin binding site of albumin. Therefore any enhancement in bilirubin fluorescence caused by the drug indicates that bilirubin is bound to the low-affinity binding sites of albumin. The use of iophenoxic acid in the determination of the extent of saturation of the high-affinity bilirubin binding site of albumin may be of value in the clinical management of infants with neonatal jaundice.     

Supersaturation with bilirubin followed by colloid formation and disposition, with a hypothesis on the etiology of kernicterus

Colloidal bilirubin-albumin particles are formed in vitro when sodium salicylate is added to a solution containing bilirubin and human serum albumin in molar ratios bilirubin/albumin less than 1:1, at pH 7.4. By determining the lower limit of salicylate concentration necessary for the aggregation as a function of the bilirubin/albumin ratio, it is found that one molecule of bilirubin is displaced from the high-affinity binding site on albumin by one molecule of salicylate, resulting in formation of colloid particles containing bilirubin and albumin in a molar proportion about 200:l. In vivo a colloidal solution of bilirubin, injected intravenously into rats, gives higher amounts of unconjugated bilirubin in liver and lungs, as compared with the amounts found after injection of an alkaline bilirubin solution. Theoretically, a process of crystalline colloid formation may be a step in the bilirubin metabolism in icterus neonatorurn. If crystallization fails to take place, supersaturation may result in high plasma levels of unconjugated bilirubin with increased risk of kernicterus. The hypothetical possibility of triggering crystallization as a preventive measure in threatening kernicterus is pointed out.