Analyses of azopigments obtained from the delta fraction of bilirubin from mammalian plasma (mammalian biliprotein).

Azopigments were obtained from the delta fraction of bilirubin (mammalian biliprotein) in cholestatic sera of men, rats and guinea pigs by diazo reaction with diazotized p-iodoaniline and analysed by t.l.c. Delta bilirubin of men and rats generated both unconjugated and glucuronide-conjugated azodipyrroles, whereas that of guinea pigs, in which the predominant form of conjugated bilirubin in serum was bilirubin monoglucuronide, generated only unconjugated azodipyrrole. We further analysed the azopigments by reversed-phase h.p.l.c. to distinguish their endovinyl and exovinyl isomers. The results indicated (a) that covalent binding of bilirubin to protein occurs exclusively on the conjugated dipyrrolic (either endovinyl or exovinyl) half of the parent conjugated bilirubin, (b) that both bilirubin monoglucuronide and bilirubin diglucuronide generate delta bilirubin, the latter yielding a 'conjugated' form of delta bilirubin that preserves the glucuronic acid moiety on the dipyrrolic half not bound covalently to protein, and (c) that therefore at least four forms of delta bilirubin exist in jaundiced sera of men and rats.     

Inaccuracies in measurement of conjugated and unconjugated bilirubin in bile with ethyl anthranilate diazo and solvent-partition methods.

A criticial evaluation was made of the ethyl anthranilate diazo and two solvent-partition methods for the determination of conjugated and unconjugated bilirubin in human and rat bile. The ethyl anthranilate diazo reagent, which reacts only with conjugated bilirubin in serum, also diazotized a variable proportion of unconjugated bilirubin in bile and thus overestimated the concentration of monoconjugates. With the Weber-Schalm and modified Folsch solvent-partition methods applied to human or rat bile, 4--9% of added 14C-labelled unconjugated bilirubin partitioned with the conjugated bilirubin in the upper phase, and 4--9% of added 14C-labelled conjugated bilirubin partitioned into the lower phase. With dog bile, the spill-over of 14C-labelled bilirubin into the lower phase was 9--11%. Analysis of azopigments from the Weber-Schalm partition confirmed that over two-thirds of the bilirubin in the lower phase represents monoconjugates, principally the less-polar monoxylosides and monoglucosides. These solvent-partition methods thus overestimate the concentration of unconjugated bilirubin in bile.     

Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal.22, 205–250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.     

Effect of cucurbitacins on bilirubin-albumin binding in human plasma

The aim of this study is to investigate the effect of three cucurbitacins (Cuc) E, D and I on the bilirubin-albumin binding, both in human serum albumin (HSA) and in plasma. Bilirubin-HSA solution and plasma free of cucurbitacins were prepared as well as others containing serial concentrations of cucurbitacins. The concentration of unbound bilirubin was determined in bilirubin-HSA solution and the direct and total bilirubin concentrations were measured in plasma (with normal or elevated bilirubinemia) by Jendrassik and Grof method. In the conditions we adopted Cuc E and D (to a lesser extent), decreased the levels of unbound bilirubin in bilirubin-HSA solution and decreased direct bilirubin concentration and total bilirubin concentration in plasma in a dose-dependent manner while Cuc I had no effect. The effect of Cuc is related to the presence of native HSA. Thus, when albumin was absent or has been denatured by heating or by urea, Cuc E did not modify bilirubin levels, suggesting that the native structure of albumin is essential for such activity. The interaction of HSA with Cuc E was investigated by fluorescence spectroscopy. Cuc E increased the intrinsic fluorescence of the protein and the magnitude of fluorescence intensity of bilirubin-albumin complex. We concluded that Cuc E and D produced a rearrangement in the structure of albumin, particularly in the domain-II, resulting in an increase in the binding of bilirubin to albumin regardless to whether it's conjugated to glucuronic acid or unconjugated.     

Anti-bilirubin monoclonal antibody. II. Enzyme-linked immunosorbent assay for bilirubin fractions by combination of two monoclonal antibodies

Among 16 monoclonal antibodies raised against covalently coupled bilirubin-bovine serum albumin, we selected two antibodies: one (designated 24G7) reacted with unconjugated and conjugated bilirubin and the other (designated 25H17) reacted only with unconjugated bilirubin. Combination of these two antibodies enabled us to determine extremely low concentrations of unconjugated and conjugated bilirubin independently by enzyme-linked immunosorbent assay (ELISA). In the assay, samples were incubated with each anti-bilirubin IgG, and then free remaining IgG was allowed to bind to the immunotiter plates coated with bilirubin-bovine serum albumin. The bound fraction of the IgG was visualized with horseradish peroxidase-conjugated rabbit anti-mouse IgG and substrate. Bilirubin concentration was determined from the absorbance at 425 nm. In this system, we could measure 10(-7)-10(-5) M unconjugated and conjugated bilirubin in samples, which is 100-fold more sensitive than Micha?lsson's diazocoupling method. The assay results gave a good correlation coefficient (0.86) compared with those determined with high performance liquid chromatography.     

Degradation of plasma bilirubin by a bilirubin oxidase derivative which has a relatively long half-life in the circulation

To enhance degradation of unconjugated bilirubin in hyperbilirubinemic subjects, we synthesized a bilirubin oxidase (EC 1.3.3.5) (BO) derivative (PEGBO) by covalently linking (2,4-bis[O-methoxy(polyethyleneglycol)]-6-chloro-s-triazine) (PEG) to the enzyme. Intravenously injected BO in rats disappeared from the circulation with a half-life of 2.5 min; the half-life of PEGBO was 190 min. Intravenously injected BO minimally and transiently decreased plasma bilirubin levels in jaundiced Gunn rats and in bile-duct-ligated jaundiced rats. In contrast, PEGBO rapidly and substantially decreased plasma bilirubin levels and the effect persisted for longer than 3 h. Renal dysfunction often occurs in patients with liver diseases. To study the role of bilirubin toxicity for the kidney, functions of transtubular transport for organic anions was measured in bile-duct-ligated jaundiced animals before and after treatment with PEGBO. Bile duct ligation decreased urinary excretion of phenolsulfophthalein (PSP), an organic anion used for renal function test. Treatment of the jaundiced animals with PEGBO increased the rate of PSP disappearance from the circulation and normalized its urinary excretion. Thus, PEGBO might be useful for the study of bilirubin toxicity in jaundiced animals.     

Excessive hepatic copper accumulation in jaundiced rats fed a high-copper diet

The response of copper metabolism to dietary copper challenge was investigated in jaundiced rats with elevated plasma concentrations of conjugated bilirubin as a result of impaired canicular transport of bilirubin glucuronides. Control and jaundiced rats were fed purified diets with either normal (64 μmol Cu/kg) or high (640 μmol Cu/kg) concentration of added copper. Copper loading produced a greater increase in hepatic copper concentrations in the jaundiced than in control rats. The greater dietary-copper-induced increase in hepatic copper in the jaundiced rats can be explained by the observed smaller rise in biliary copper excretion and a greater efficiency of dietary copper absorption. In individual rats, there was a positive relationship between hepatic copper concentrations and biliary copper concentrations. It is suggested that not the transport of copper from liver cells to bile but that from plasma to bile is diminished in the jaundiced rats. The elevated plasma copper concentrations in the jaundiced rats may support this suggestion.     

A novel method to determine total and free serum bilirubin.

The determination of total (unconjugated) and free serum bilirubin concentrations using a novel and sensitive method based on static fluorescence quenching of daneyl bovine serum albumin was developed. The method allowed the use of a sample of 5 μl or less to determine total bilirubin over a range of 10–200 μg/ml with good recovery (94.9 ± 2.2%). For the determination of total bilirubin, a denaturation medium containing 8 m urea, 10 mm dithloerythreitol, and 0.1 m Tris was employed to eliminate interference by human serum albumin itself. The method was also tested with patients' sera containing negligible conjugated bilirubin in order to compare it to a commonly used “diazo” method. The correlation between the two methods gave a practically linear relation (γ = 0.99). The effects of a number of potentially interfering substances were tested and the results showed the test was specific for bilirubin. Concentrations of free bilirubin were determined without adding a denaturation agent. The experimental values were in agreement with those calculated theoretically using the isotherm of a single binding site and an association constant of human serum albumin to bilirubin of 1.5 × 10⁸m^?1.     

Bilirubin UDP-glucuronyltransferase activity in human fetal liver homogenates]

The bilirubin UDP GT-activity of the liver was studied from week 15 of pregnancy in 23 human fetuses and mature and immature dead newborns, respectively. No measurable bilirubin UDP GT-activity was found up to week 26 of pregnancy. The development of enzymatic activity presumably starts only postnatally, irrespective of gestational age of birth weight. Up to the fifth day the bilirubin UDP GT-activity is less than 0.2 mg of conjugated bilirubin/g liver/h, reaching in the second week values that correspond to approximately 20--30% of adult values. This activity is sufficient to prevent a clinically important hyperbilirubinemia because the concentration of unconjugated serum bilirubin was in all children below 12mg/100 ml.     

Medroxyprogesterone acetate in human serum

Conjugates of medroxyprogesterone acetate (MPA) in human serum are investigated using chromatography and techniques (equilibrium dialysis, gel filtration, and polyacrylamide gel electrophoresis) previously described for studying the binding of MPA. 17 serum samples were obtained from 7 women at various times after the intramuscular injection of 150 mg Depo-Provera. Mean concentration of MPA in the unconjugated fraction of serum was 3.9 mg/ml (range 0.8-10.7 ng/ml); in the conjugated fraction, the value was 2.7 ng/ml (range 0.6-11.4 ng/ml), a mean value of 81.7% (range 18.4-286%) of that in the unconjugated fraction. The conjugate appears to be mainly a glucuronide since solvolysis released only small amounts of MPA. MPA metabolites were also detected in blood. The MPA levels in blood measured by radioimmunoassay were generally lower when serum was extracted with an organic solvent rather than when the assay was carried out directly in the serum. This finding suggests the presence in blood of either MPA in a conjugated form or metabolites interacting with the antiserum which were not extracted by the solvents used. Equilibrium dialysis showed that undiluted plasma bound 85.8% of triated hydrogen-MPA; with increasing dilution of the plasma, the amount of bound triated hydrogen-MPA decreased. The apparent association constant calculated according to the method of Vermeulen and Verdonck was 2.6 x 10 4 1/mol. MPA appeared to be loosely bound to albumin in blood but there was no specific binding protein for the steroid. MPA conversion to the glucuronide may be 1 of the factors regulating the level of the unconjugated but presumably biologically active steroid in blood.     

The effect of bilirubin photoisomers on unbound-bilirubin concentrations estimated by the peroxidase method.

Unbound bilirubin is oxidized to nearly colourless substances in the presence of H2O2 or ethyl hydroperoxide and horseradish peroxidase. To predict the risk of kernicterus (degenerated yellow pigmentation of nerve cells), this principle has been widely utilized for estimating the concentration of unbound bilirubin in hyperbilirubinaemic serum. However, the serum contains polar geometric photoisomers of bilirubin. Therefore, to clarify the effect of bilirubin photoisomer concentrations on unbound-bilirubin concentration, the concentration of bilirubin and its photoisomer and of unbound bilirubin in samples obtained from experiments in vivo and in vitro were simultaneously and individually estimated by h.p.l.c. and the peroxidase method. During photoirradiation, both in vivo and in vitro, the serum polar (ZE)-bilirubin IX alpha concentration increased remarkably, but unbound-bilirubin values were not affected at all. However, during experiments in vitro, unbound bilirubin concentrations increased only when concentrations of the rather polar (EZ)- and (EE)-cyclobilirubin IX alpha increased considerably in a human serum albumin-bilirubin solution irradiated with blue light. Thus it is concluded that unbound-bilirubin concentrations, and consequently the initial rate of the peroxidase reaction, is not accelerated by the increase in either (ZE)-bilirubin or (EZ)-cyclobilirubin concentration within the clinically observed range.     

Demonstration of Conjugated Dopamine in Monkey CSF by Gas Chromatography-Mass Spectrometry

Abstract: A method for measuring unconjugated and conjugated dopamine in body tissues and fluids is described. Conjugated dopamine was hydrolyzed in acid to unconjugated dopamine, separated from the sample matrix by alumina chromatography, and assayed by gas chromatography-mass spectrometry. Conjugated dopamine was detected in greater concentrations than unconjugated dopamine in CSF taken from lateral ventricle or thecal sac of the Rhesus monkey. Haloperidol administration did not increase the levels of conjugated dopamine in lumbar CSF.     

Diagnosis of Gilbert's Syndrome: Role of Reduced Caloric Intake Test

Reduction in caloric intake to 400 a day for 72 hours resulted in a significant increase in the plasma bilirubin concentration in patients with Gilbert''s syndrome and in normal subjects. This was due to an increase in unconjugated pigment. There was no significant increase in the bilirubin concentration in patients with liver disease or haemolytic anaemia.The increase in unconjugated bilirubin was signficantly greater in Gilbert''s syndrome than in normals but only when the initial bilirubin concentration was raised. It was usually seen within 24 hours of reducing the caloric intake. An increase of 100% or more suggests that unconjugated hyperbilirubinaemia is due to Gilbert''s syndrome. In the normal subjects the unconjugated bilirubin level did not exceed 1·0 mg/100 ml.The increase in unconjugated bilirubin concentration on reducing the caloric intake may be due to decreased hepatic bilirubin uridine diphosphate glucuronyl transferase activity, which was shown to be present in seven rats starved for 72 hours. The effect of a 400 calorie diet for 24 hours on the unconjugated bilirubin level may distinguish Gilbert''s syndrome from other causes of unconjugated hyperbilirubinaemia.     

Enzymatic oxidation of unconjugated bilirubin to assess its interactions with taurocholate

The rate of peroxidation of unconjugated bilirubin (UCB), catalyzed by horseradish peroxidase (HRP), has been employed by Jacobsen (1969. FEBS Lett. 5: 112-114) to assess the fraction of unbound UCB in the presence of serum albumin. We used this method to examine the interactions of UCB with taurocholate (TC) at pH 8.2, assuming solubilization of UCB by TC is due to pigment binding and/or to partitioning into the micelle, thus rendering UCB unavailable for peroxidation. Inhibition of UCB peroxidation conformed with predictions based on these assumptions and demonstrated significant interaction of UCB with both monomeric and micellar TC. Although significant inhibition of UCB peroxidation was seen with TC monomer, inhibition was even greater with TC micelles. In contrast, pyrogallol, another substrate of HRP, acted very differently in the presence of TC. Inhibition of pyrogallol peroxidation by TC was much less than with UCB and occurred primarily with monomeric TC, with little further inhibition in the micellar range. The results of this study suggest that at taurocholate concentrations above 50 mM, similar to the physiologic bile salt concentrations in human bile, at least 99% of UCB is bound to bile salt, dramatically decreasing the concentration of unbound UCB. Since bile salts also bind Ca2+, they play a dual role in protection against the precipitation of calcium bilirubinate from bile. Therefore, bile salts are a major factor in the prevention of the formation and growth of pigment gallstones.     

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.     

Development and validation of a method to determine the unbound paclitaxel fraction in human plasma

Paclitaxel is pharmaceutically formulated in a mixture of Cremophor EL and ethanol (1:1, v/v). The unbound fraction of the anticancer drug paclitaxel in plasma is dependent on both plasma protein binding and entrapment in Cremophor EL micelles. We have developed a simple and reproducible method for the quantification of the unbound paclitaxel fraction in human plasma. Human plasma was spiked with [3H]paclitaxel and [14C]glucose (unbound reference) and incubated at 37 degrees C for 30 min. Plasma ultrafiltrate was prepared by a micropartition system (MPS-1) and collected in a sample cup containing 100 microl of plasma to prevent the loss of paclitaxel due to adsorption. The radionuclides were separated after combustion of the biological samples using a sample oxidizer and the radioactivity was determined by liquid scintillation counting. The unbound fraction of paclitaxel was calculated by dividing the ratios of 3H and 14C in plasma ultrafiltrate and in plasma. The method was thoroughly validated using human plasma spiked with pharmacologically relevant concentrations of paclitaxel (10-1000 ng/ml) and Cremophor EL (0.25-2.0%). The method was precise, with a within-day precision ranging from 3.9 to 11.0% and a between-day precision ranging from 5.8 to 13.1%. In patient plasma with low serum albumin values containing 1% of Cremophor EL, the unbound fraction appeared to be significantly higher than that in plasma with normal albumin values. The determination of the unbound fraction of paclitaxel proved to be stable during a 10-week storage at -20 degrees C. Furthermore, the assay was applicable in patient samples. This assay can be used to determine the unbound fraction of paclitaxel in plasma. Moreover, its design should allow the determination of the unbound concentrations of other hydrophobic drugs.     

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.     

A new tactic for the treatment of jaundice: an injectable polymer-conjugated Bilirubin oxidase

Bilirubin oxidase (BOX) derived from Myrothecium verrucaria was modified with polyethylene glycol (PEG). When the conjugated PEG-BOX was given intravenously to rats, its plasma half-life was 20 times longer than that of native BOX. In our preliminary investigations with experimentally jaundiced rats, the plasma bilirubin level dropped to normal after only one injection, and the low bilirubin level could be maintained for 12-48 hr; native BOX had a transitory suppressive effect that lasted only a few hours. The antigenicity of PEG-BOX was greatly reduced as expected. PEG-BOX appears to have potential value for the treatment of hyperbilirubinemia observed in such diseases as fulminant hepatitis and neonatal bilirubin encephalopathy.     

Molecular basis of bilirubin-induced neurotoxicity

Unconjugated bilirubin (UCB), at slightly elevated unbound concentrations, is toxic to astrocytes and neurons, damaging mitochondria (causing impaired energy metabolism and apoptosis) and plasma membranes (causing oxidative damage and disrupting transport of neurotransmitters). Accumulation of UCB in the CSF and CNS is limited by its active export, probably mediated by MRP1/Mrp1 present in choroid plexus epithelia, capillary endothelia, astrocytes and neurons. Upregulation of MRP1/Mrp1 protein levels by UCB might represent an important adaptive mechanism that protects the CNS from UCB toxicity. These concepts could explain the varied susceptibility of newborns to bilirubin neurotoxicity and the occurrence of neurological damage at plasma UCB concentrations well below therapeutic guidelines, and are relevant to the increasing prevalence of bilirubin encephalopathy in newborns.     

The Biological Effects of Bilirubin Photoisomers

Although phototherapy was introduced as early as 1950’s, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.