Photoactivated covalent binding of [3H]bilirubin to human serum albumin.

A one-step procedure has been developed for the preparation of [3H]bilirubin IX-alpha in good yield from unlabelled bilirubin. Irradiation of an aqueous solution of [3H]bilirubin IX-alpha in the presence of human serum albumin results in the covalent attachment of the bilirubin to the protein. Preliminary degradation studies have been carried out to locate the site of attachment of the bilirubin to the albumin.     

The enzyme-catalyzed formation of bilirubin diglucuronide by a solubilized preparation from cat liver microsomes

When bilirubin monoglucoronide is incubated with a preparation from the 105 000 × g-supernatant of deoxycholate-treated cat liver microsomes, bilirubin diglucuronide is formed. This is an UDPglucuronate-dependent reaction whereby bilirubin IXα monoglucuronide is stoichiometrically converted into bilirubin IXα diglucuronide.The pH optimum for the conversion of bilirubin into bilirubin monoglucuronide lies between pH 8.0 and pH 8.8. For the conversion of mono- into diglucuronide two optima were found, one at about pH 6.5 and another at pH 8.1.When incubation was performed at pH 6.5 and the enzyme protein concentration was lowered, bilirubin monoglucuronide started to isomerise. As a result of this isomerisation bilirubin diglucuronide is also formed. Diglucuronide formation according to this mechanism however, can be clearly differentiated from the enzyme-catalyzed diglucuronide formation.By the formation of bilirubin monoglucuronide, one monoglucuronide isomer is preferentially synthesized.The alkaline-labile bilirubin conjugates in the bile of cats and rats have mainly the IXα isomeric structure. This suggests that in these animals bilirubin diglucuronide is formed enzymically as the bilirubin moiety of diglucuronide, formed by means of the isomerisation reaction, has predominantly the XIIα structure.     

Bilirubin diffusion through lipid membranes

The possibility that bilirubin can diffuse through lipid bilayers is investigated with liposomes prepared from dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (egg PC) with 22 mole percent cholesterol, and a lipid extract preparation from N115 neuroblastoma cells. Liposomes were prepared with internalized bilirubin and bovine or human serum albumin, and bilirubin efflux into an exogenous solution of human serum albumin was measured. Efflux from DPPC liposomes was significantly higher above the phase transition temperature than below it. This change was dependent on the lipid undergoing a phase transition and could not be accounted for by 6 K change in temperature. Maximum bilirubin efflux from egg PC-cholesterol liposomes was found to depend on the relative internal and external albumin pools, suggesting an equilibrium distribution of bilirubin between them. These observations demonstrate that bilirubin can diffuse freely through these lipid membranes.     

The formation of bilirubin and p-nitrophenyl glucuronides by rabbit liver

1. Glucuronide formation of bilirubin and p-nitrophenol in vitro with excess of UDP-glucuronic acid by UDP-glucuronyltransferase from livers of young and adult rabbits was studied. 2. The development of UDP-glucuronyltransferase for the two substrates followed a markedly different pattern during maturation of young rabbits, p-nitrophenol-conjugation ability being much higher at birth than that for bilirubin. 3. Mg(2+) increased bilirubin conjugation, but inhibited p-nitrophenyl glucuronide formation. 4. p-Nitrophenol acted as a potent non-competitive inhibitor for bilirubin conjugation but bilirubin did not affect p-nitrophenyl glucuronidation. 5. The enzyme for bilirubin conjugation was inactivated at pH9 during treatment with snake venom, whereas in the same preparation the activity of the corresponding enzyme for p-nitrophenol was enhanced. In addition, some solubilization of the latter enzyme could be achieved by this method. 6. The possibility of the existence of more than one enzyme system for the formation of O-glucuronides is discussed.     

耐高温重组CotA蛋白的胆红素氧化酶反应动力学及活性鉴定

已知源于枯草芽孢杆菌内生孢子的CotA蛋白具有漆酶和胆红素氧化酶活性。然而,其分离纯化极为困难。本研究对表达与纯化的重组CotA蛋白的胆红素氧化酶特性及氧化还原功能进行鉴定。基因转染及筛选获得了表达CotA的P. pastoris菌株|继而,表达的重组CotA蛋白经DEAE-Sepharose FF 及Sephadex G-75层析分离与纯化,产物得率为25%,纯化产物的酶比活性为 4 U/mg。经SDS-PAGE 和 MALDI-TOF MS 分析显示,其分子质量为65 kD。纯化的CotA蛋白能够催化胆红素氧化,生成胆绿素,且催化反应速率受反应溶液中溶解氧含量的影响,提示纯化的重组CotA具有胆红素氧化酶活性。酶反应进一步证明,CotA的胆红素氧化酶反应最适pH值为pH 8.0,最适温度为60℃。该酶在90℃条件下的半衰期为7 h,提示CotA胆红素氧化酶具有高度的热稳定性。CotA修饰的摄谱仪石墨电极可直接电催化分子氧（O2）还原,具有很好的电流响应。我们的结果表明,重组的CotA蛋白具有耐高温胆红素氧化酶活性。更重要的是,我们的结果还提示重组的CotA蛋白在酶生物燃料电池阴极的制备上具有较好的应用潜能。     

Formation of bilirubin glucoside

1. Rat liver microsomal preparation can effect the transglucosylation from UDP-glucose to bilirubin in the presence of Mg(2+). 2. Other nucleotides, namely CDP-glucose, ADP-glucose and GDP-glucose, were not active as glucosyl donors. 3. Only trace amounts of galactose, galacturonic acid and N-acetylglucosamine were conjugated to bilirubin when their respective UDP derivatives were used in the reaction mixture. 4. The azobilirubin glucosides produced by coupling with p-diazobenzenesulphonic acid and diazotized ethyl anthranilic acid were separable from the corresponding azobilirubin glucuronides by t.l.c. 5. The glucoside was, however, hydrolysed by both beta-glucosidase and various preparations of beta-glucuronidase; azobilirubin and glucose were liberated in the process. 6. Kinetic studies showed that the effects of pH and Mg(2+) on the two conjugating systems were similar. 7. The specific activities of hepatic bilirubin UDP-glucosyltransferase, expressed as mug of bilirubin ;equivalents' conjugated/h per mg of protein, are respectively 1.7 and 2.4 for male and female rats. 8. The K(m) values for bilirubin and UDP-glucose are 5.7x10(-5)m and 1.6x10(-3)m respectively. 9. The glucoside and glucuronide conjugations of bilirubin are discussed in relation to the availability of the conjugating agents and aglycone in the liver.     

A polypeptide conjugate of bilirubin from human bile.

An alkali-stable bilirubin conjugate has been obtained from human T-tube bile as its phenylazo derivative. The conjugate consists of a polypeptide probably of molecular weight 7000 to which the azo pigment of bilirubin is linked covalently through its carboxyl group. It thus constitutes the first biliprotein found in mammals. It is not known whether both carboxyl groups of native bilirubin participate in the binding of the conjugating protein, nor has it been possible to determine the number of pigment moieties occurring on a single polypeptide chain. The isolation makes use of the tendency of the conjugate to form large aggregates and involves the following steps: azo coupling of the native bile, (NH4)2S04 precipitation of macromolecules and aggregates, removal of low molecular weight contaminants by dialysis and gel filtration (first on Sepharose 6B IN 6 M guanidine, then on Sephadex LH-20 in 50% acqueous 2-chloroethanol) and a concluding purification by chromatography on p-aminobenzyl cellulose using a PH gradient. The final preparation appeared to be homogeneous on polyacrylamide electrophoresis.     

Isolation and properties of conjugated bilirubin from bile

1. A simple, rapid solvent partition method is described for isolation of conjugated bilirubin, free of unconjugated bilirubin, bile salts, phospholipids and cholesterol, from rat bile. Yields are 40-58%. The product is a phosphate-buffered solution containing approx. 0.4mg of bilirubin/ml, principally as mono- and di-glucuronide conjugates. The method may be modified for isolation of conjugates from human bile with 15-22% yield, and for preparation of unconjugated bilirubin from rat or human bile with yields of 55-62%. 2. The conjugated pigment has red-brown fluorescence and an absorption maximum at 450nm with in(mM) 59.8cm(-1). Diazotization by the Malloy-Evelyn method gives a direct Van den Bergh reaction (in water) 12% greater than the total reaction (in methanol), with in(total) 28.4x10(3)lmol(-1)cm(-1) at 550nm. After desalting by elution from Sephadex LH-20 in 50% (v/v) ethanol, the product gave water-soluble mustard-yellow crystalline needles. Such desalted conjugates were precipitated by Pb(2+) but not by Ba(2+), Ca(2+) or Zn(2+). 3. At pH7.0 and 37 degrees C the conjugated bilirubin was oxidized at a rate of 1%/h without hydrolysis, whereas 84% was hydrolysed by beta-glucuronidase or aqueous alkali. 4. Mono- and di-glucuronides were separated by elution from Sephadex LH-20 in 95% (v/v) ethanol or by extraction with chloroform at pH3.2-3.4. The monoconjugated bilirubin did not become labelled during incubation with unconjugated [(14)C]bilirubin, and chromatographed as a single spot without dissociating into unconjugated bilirubin and diglucuronide as would be expected of a complex. 5. After intravenous injection of mono- or di-conjugated [(14)C]bilirubin into normal or Gunn rats, 79-91% was excreted in bile and 2-7% in urine over 2h. In these experiments injected diglucuronide was not hydrolysed whereas 30-41% of injected monoglucuronide was converted into diglucuronide by the normal but not by the Gunn rats. The evidence favours the existence of a true bilirubin mono-glucuronide that is not a complex.     

The effect of steroids and nucleotides on solubilized bilirubin uridine diphosphate glucuronyltransferase

1. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis-Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The K(m) for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca(2+) were inhibitory in the absence of Mg(2+) but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3beta-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis.     

Photobilirubin II

An improved preparation of photobilirubin II in ammoniacal methanol is described. Evidence is presented which distinguishes between the two structures proposed earlier for photobilirubin II in favour of the cycloheptadienyl structure. Nuclear-Overhauser-enhancement measurements with bilirubin IX alpha and photobilirubin II in dimethyl sulphoxide are complicated by the occurrence of negative and zero effects. The partition coefficient of photobilirubin II between chloroform and phosphate buffer (pH 7.4) is 0.67.     

微生态制剂（培菲康）治疗新生儿高胆红素血症临床疗效评价

目的评价微生态制剂（培菲康）治疗新生儿高胆红素血症的临床疗效。方法110例在福建医科大学附属第一医院儿科住院治疗的新生儿高胆红素血症患儿分为治疗组和对照组;对照组65例给予肝酶诱导剂、光疗等综合治疗;治疗组45例在综合治疗基础上加用培菲康治疗。治疗前后检测患儿血清总胆红素和间接胆红素含量。采用SPSS11.0软件包进行统计学分析,α=0.05为显著性检验标准。结果时间-效应分析显示培菲康治疗组和对照组血清总胆红素下降值及95%CI（μmol/L）分别为142.6（126.7～158.4）、74.1（66.9～81.3）,血清间接胆红素下降值及95%CI（μmol/L）为115.7（103.3～128.1）、62.6（53.7～71.7）,差异有显著性（P〈0.05）;多元Cox回归分析显示患儿年龄、体重、孕周、入院前病程、入院血清总胆红素值/间接胆红素值及应用培菲康治疗为影响高胆红素血症患儿血清总胆红素/间接胆红素下降的主要因素;治疗组和对照组治愈例数（治愈率）分别为37例（82.2%）、8例（17.8%）,好转例数（好转率）分别为47例（72.3%）、10例（27.7%）,χ^2=1.448,P=0.229。2组均未见明显不良反应发生。结论应用培菲康治疗新生儿高胆红素,可促进胆红素分解和排泄,减少肠肝循环,减少肠道重吸收未结合胆红素,是治疗新生儿高胆红素血症一种可靠、安全的药物。     

Bilirubin glucuronyltransferase. Specific assay and kinetic studies

1. Bilirubin glucuronide was synthesized in vitro in a system containing a rat liver microsomal fraction, UDP-glucuronic acid, Mg(2+) and bilirubin. The enzymic synthesis was accomplished without the addition of a bilirubin carrier. 2. Azobilirubin and azobilirubin glucuronide were separated by t.l.c. and paper chromatography and the measurement of the conjugate provided a specific assay for bilirubin UDP-glucuronyltransferase (EC 2.4.1.17). 3. This diazo compound was labelled when [U-(14)C]UDP-glucuronic acid was employed in the transglucuronidation reaction. 4. Identity of the glucuronide nature of the product was further confirmed by hydrolysis with beta-glucuronidase prepared from limpets and Helix pomatia. In each instance azobilirubin and glucuronic acid were liberated. 5. There was a close correlation between the bilirubin glucuronyl-transferase activity as measured by two procedures, colorimetric and radioisotopic. The specific activities so measured were 19nmol of bilirubin ;equivalents' conjugated/h per mg of protein and 16.9-18.4nmol of UDP-glucuronic acid incorporated/h per mg of protein, respectively. On this basis, it was concluded that the major product formed in vitro was bilirubin monoglucuronide; this represents about 77% of the total products formed. 6. The K(m) values for bilirubin and UDP-glucuronic acid at pH8.2 are 3.3x10(-4)m and 1.67x10(-3)m, respectively. 7. The addition of Mg(2+) at a final concentration of 5mm to the reaction mixture increased the rate of conjugation by 5.6-fold in the microsomal preparation that had been subjected to overnight dialysis against 10mm-EDTA (disodium salt). 8. Diethyl-nitrosamine at a final concentration of 1-20mm has no effect on the glucuronidation of bilirubin in vitro.     

Advances in the Theory and Practice of Exchange Transfusions

In the management and prevention of hemolytic disease of the newborn, exchange transfusions seem destined to remain the mainstay of therapy for some time to come.Our current knowledge of bilirubin metabolism has altered the indications for the procedure and introduced such useful new adjuncts to therapy as albumin infusions. The decision to do an exchange transfusion cannot be made by any one rule, but must be individualized for each patient and take into account all the factors known to influence the risks of bilirubin toxicity and the exchange procedure. A thorough evaluation of the infant''s condition, particularly his cardiorespiratory and metabolic status (including blood pH, gas and albumin determinations), will provide valuable information as a guide to therapy. The limited capacity of some newborn infants to make adequate physiological adaptations to a variety of stresses imposed by the procedure influences the preparation of donor blood, the rate and volume of exchange and the time at which it should be done.A clear understanding of the mechanics of the exchange and the distribution of indirect bilirubin within the body will permit more accurate prediction of what can be accomplished in bilirubin removal and correction of hematocrit with exchanges of different volumes. When weighing the risk of kernicterus against that of exchange transfusion, the experience of the operator and the availability of suitable facilities cannot be ignored.     

Effect of alpha-lipoic acid preparations on bilirubin and transferrin levels and ferritin iron and transferrin iron content

The influence of ethylendiamine salt of alpha-lipoic acid on the indices of iron metabolism in patients with occupational pathologies has been studied using quantitative electron spin resonance spectroscopy. In the cases of treatment in the patients suffering from hyperferremia the decrease in transferrin iron concentration in the whole blood and plasma occurs correlating with the enhancement of iron excretion from urine and decline of bilirubin level in serum. We have found that the preparation chelates iron from iron (III)-citrate complex and form stable iron (III) complexes. The conclusion is that the positive effects of lipoic acid preparation in the patients with hyperferremia at least partially could be associated with normalization of iron exchange and reduction in the labile iron pool.     

Principles of a competitive binding assay for the study of the interactions of poorly water-soluble ligands with their soluble binding partners. Application to bilirubin with the use of Sephadex G-10 as a competitive adsorbent.

1. A generally applicable method is described for obtaining experimental data on the interactions between a poorly water-soluble ligand and soluble binding factors, with the use of chemically inert solid adsorbent. The equilibrium distribution of the ligand between the liquid phase containing the soluble binders and the adsorbent must be measured and knowledge of the binding isotherm of the adsorbent is required. Procedures are given for the calculation of the binding parameters. 2. The method has been applied to quantify the interactions of bilirubin with serum and liver cytosol from the rat, Sephadex G-10 serving as the competing adsorbent. Reversible adsorption keeps the concentration of the free ligand low, thereby preventing formation of colloidal bilirubin. The sensitivity of the procedure accommodates the rather high binding affinities by which bilirubin generally interacts with its specific binding proteins. 3. The binding activities of serum and liver cytosol are of comparable magnitude. Binding of bilirubin by rat serum can be described by two independent binding sites, the affinities of which differ by two orders of magnitude. Only the site with the higher affinity appears to be of physiological importance. The major bilirubin-binding sites of rat liver cytosol seem to contribute equally to the overall binding activity of this preparation, provided that GSH is present.     

Separation and structural analysis of vinyl- and isovinyl-azobilirubin derivatives

The coupling reaction of bilirubin with the diazonium salts of ethyl anthranilate or of aniline yields two isomeric azopigments. These can be separated by t.l.c. as their methyl esters. The mass spectra of each pair of azopigments are very similar, showing that they are isomers. Proton-magnetic-resonance spectrometric studies show that they differ in the positions of the substituents on the pyrrolenone end ring; in one compound the methyl and vinyl groups are interposed compared with the other compound. These azo compounds were used as reference standards for determination of the site of conjugation in bilirubin monoglucuronide prepared enzymically. Analysis showed that conjugation occurs at the carboxyethyl side chain of both sides of the bilirubin molecule. During the preparation of the ethyl anthranilate reference compounds a series of minor azopigments were isolated by t.l.c. Analysis of the mass spectra of many of these showed that three side reactions can occur: (1) methylation of the imide carbonyl group; (2) addition of methanol or water to the vinyl substituent; (3) transmethylation of the ethoxycarbonyl group.     

Enzymatic conversion of bilirubin monoglucuronide to diglucuronide by rat liver plasma membranes.

Formation of bilirubin monoglucuronide from unconjugated bilirubin requires a microsomal enzyme, UDP-glucuronate glucuronyltransferase (EC 2.4.1.17). Conversion of bilirubin monoglucuronide to bilirubin diglucuronide, the major bilirubin conjugate in bile, was studied in subcellular fractions of rat liver. The highest specific activity for bilirubin diglucuronide formation occurred in a fraction highly enriched in plasma membranes. Studies of reaction stoichiometry and utilization of UDP-D-[14C]glucuronic acid revealed that conversion of bilirubin monoglucuronide to bilirubin diglucuronide is not catalyzed by UDP-glucuronyltransferase, and results from transglucuronidation of bilirubin monoglucuronide, with formation of bilirubin diglucuronide and unconjugated bilirubin. When unconjugated bilirubin was infused intravenously into rats at rates exceeding the maximal hepatic excretory capacity, bilirubin monoglucuronide accumulated in serum and bilirubin diglucuronide was found exclusively in bile as the predominant bilirubin metabolite. These results suggest that formation of bilirubin diglucuronide occurs at the surface membrane of the liver cell. Conversion of bilirubin monoglucuronide to bilirubin diglucuronide may play a role in the transport of bilirubin glucuronides from liver to bile.     

Measurement of plasma unbound unconjugated bilirubin

A method is described for measuring the unconjugated fraction of the unbound bilirubin concentration in plasma by combining the peroxidase method for determining unbound bilirubin with a diazo method for measuring conjugated and unconjugated bilirubin. The accuracy of the unbound bilirubin determination is improved by decreasing sample dilution, eliminating interference by conjugated bilirubin, monitoring changes in bilirubin concentration using diazo derivatives, and correcting for rate-limiting dissociation of bilirubin from albumin. The unbound unconjugated bilirubin concentration by the combined method in plasma from 20 jaundiced newborns was significantly greater than and poorly correlated with the unbound bilirubin determined by the existing peroxidase method (r = 0.7), possibly due to differences in sample dilution between the methods. The unbound unconjugated bilirubin was an unpredictable fraction of the unbound bilirubin in plasma samples from patients with similar total bilirubin concentrations but varying levels of conjugated bilirubin. A bilirubin-binding competitor was readily detected at a sample dilution typically used for the combined test but not at the dilution used for the existing peroxidase method. The combined method is ideally suited to measuring unbound unconjugated bilirubin in jaundiced human newborns or animal models of kernicterus.     

Multiplicity of heme oxygenase isozymes. HO-1 and HO-2 are different molecular species in rat and rabbit

We report on the detection and characterization of two forms of heme oxygenase in rabbit tissues and provide data suggesting that heme oxygenases in rat and rabbit are not identical and constitute a group of heterogenous proteins. Certain molecular properties, however, are shared by the isozymes in rat and rabbit; the predominant form of the enzyme in control liver and testis is HO-2, in the liver HO-1 is the inducible form, and in the brain HO-1 is not detectable. HO-1 was purified from liver of rabbits treated with bromobenzene to near homogeneity with a specific activity of 8,270 nmol of bilirubin/mg/h and compared with a homogenous preparation of rat HO-1 with a specific activity of 6,220, also obtained from bromobenzene-treated animals. Rat and rabbit HO-1, on sodium dodecyl sulfate-polyacrylamide gel, had molecular weights of 30,000 and 30,700, respectively. Rabbit HO-2 was partially purified from testis to a specific activity of 386 nmol of bilirubin/mg/h and compared with a purified preparation of rat testis HO-2 with a specific activity of 5,700. Using Western immunoblotting, rabbit HO-2 displayed intense cross-reactivity with antibody raised in rabbit to sodium dodecyl sulfate-denatured rat HO-2, and had a substantially larger molecular weight than the rat HO-2 (42,000 versus 36,000). Rabbit HO-1 did not cross-react with antibody to rat HO-1 which was also raised in rabbit. Unlike the rat enzymes, rabbit HO-1 and HO-2 did not differ in thermolability. It is speculated that HO-1 in rat and rabbit, and possibly HO-2, have evolved from divergent evolution of a common ancestral gene(s).     

Glutathione S-transferase from beef heart: structural and kinetic properties

A simple and rapid method for the purification of glutathione S-transferase is described. The physical and kinetic properties of purified enzyme are reported. The protein is constituted of two identical subunits with a total molecular weight of 46,000 daltons. The isoelectric focusing of crude cytosol or purified preparation gives a single peak of activity with a pI of 7.1. The kinetic analysis shows a relatively strict substrate specificity. Only 1-chloro-2,4-dinitrobenzene is conjugated to reduced glutathione at an appreciable rate. The peroxidase activity of the enzyme with respect to cumene hydroperoxide as substrate is negligible. Hemin and bilirubin are competitive inhibitors of catalytic activity.