Karel P.M. Heirwegh

Unconjugated Bilirubin and an Increased Proportion of Bilirubin Monoconjugates in the Bile of Patients with Gilbert's Syndrome and Crigler-Najjar Disease

Bilirubin pigments were studied in the bile of 20 normal adults, 25 patients with Gilberts syndrome, 9 children with Crigler-Najjar disease, and 6 patients with hemolysis, to determine how a deficiency of hepatic bilirubin UDP-glucuronosyltransferase would affect the end products of bilirubin biotransformation. In the bile from patients with Gilberts syndrome, a striking increase was found in the proportion of bilirubin monoconjugates (48.6+/-9.8% of total conjugates) relative to that in normal bile (27.2+/-7.8%). This increase was even more pronounced in children with Crigler-Najjar disease, in whom, even in the most severe cases, glucuronide could always be demonstrated in the bile. Furthermore, unconjugated bilirubin-IXalpha was unquestionably present in the bile of these children and amounted to 30-57% of their total bilirubin pigments (<1% in the controls). It was not possible to predict from the biliary bilirubin composition whether a child would respond to phenobarbital therapy or not. Bile composition was normal in patients with hemolysis, except when there was associated deficiency of hepatic glucuronosyltransferase. Therefore, the observed alterations were not a simple consequence of unconjugated hyperbilirubinemia. The present findings suggest that Crigler-Najjar disease represents a more pronounced expression than Gilberts syndrome of a common biochemical defect. Hepatic bilirubin UDP-glucuronosyltransferase deficiency leads to decreased formation of diconjugates with an ensuing increase in the proportion of bilirubin monoconjugates in bile; in the most severe cases, an elevated content of biliary unconjugated bilirubin is also found.

Chromatographic analysis and structure determination of biliverdins and bilirubins

Recent applications of thin-layer chromatographic (TLC) and high-performance liquid chromatographic (HPLC) procedures has revealed an unexpected wide variety of naturally occurring unconjugated and conjugated bilirubins. Biliverdins seems to occur only in unconjugated forms, mainly as the IX alpha isomer. Several synthetic biliverdins and bilirubins present interesting models for biochemical and metabolic studies. Owing to recent recognition of the astounding heterogeneity of natural bilirubins and to the various artifactual changes that bile pigments can undergo, considerable confusion has existed, and still exists, with regard to the nomenclature of the bile pigments and their derivatives. To set a background for further discussion, the present review starts with a brief discussion of nomenclature and of the various characteristic forms of lability of the bile pigments. TLC and HPLC procedures for preparation and analysis of unconjugated biliverdins and bilirubins and their methyl ester and sugar ester conjugates, as well as procedures for analysis of bilirubin-protein conjugates, are then discussed. Since, in view of the lability and pronounced heterogeneity of bile pigments, it is important to assess the composition and nature of chromatographically isolated pigments, the review is concluded by a brief evaluation of various structural tests.

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX☆

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beers law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.

Uridine diphosphate-glucuronic acid-independent conversion of bilirubin monoglucuronides to diglucuronide in presence of plasma membranes from rat liver is nonenzymic.

TWO ROUTES HAVE BEEN PROPOSED FOR CONVERSION OF BILIRUBIN MONOGLUCURONIDE TO THE DIGLUCURONIDE: glucuronyl transfer (a) from UDP-glucuronic acid to bilirubin monoglucuronide, catalyzed by a microsomal UDP-glucuronyltransferase, and (b) from one molecule of bilirubin monoglucuronide to another (transglucuronidation), catalyzed by an enzyme present in liver plasma membranes. The evidence regarding the role of the latter enzyme for in vivo formation of bilirubin diglucuronide is conflicting. We therefore decided to reexamine the transglucuronidation reaction in plasma membranes and to study the conversion of bilirubin monoglucuronide to diglucuronide in vivo. Purified bilirubin monoglucuronide was incubated with homogenates and plasma membrane-enriched fractions from liver of Wistar and Gunn rats. Stoichiometric formation of bilirubin and bilirubin diglucuronide out of 2 mol of bilirubin monoglucuronide was paralleled by an increase of the IIIalpha- and XIIIalpha-isomers of the bilirubin aglycone, thus showing that dipyrrole exchange, not transglucuronidation, is the underlying mechanism. Complete inhibition by ascorbic acid probably reflects intermediate formation of free radicals of dipyrrolic moieties. The reaction was nonenzymic because it proceeded independently of the protein concentration and heat denaturation of the plasma membranes did not result in decreased conversion rates. Collectively, these findings show spontaneous, nonenzymic dipyrrole exchange when bilirubin monoglucuronide is incubated in the presence of rat liver plasma membranes. Because bilirubin glucuronides present in biological fluids contain exclusively the bilirubin-IXalpha aglycone, formation of the diglucuronide from the monoglucuronide by dipyrrole exchange does not occur in vivo. Rapid excretion of unchanged bilirubin monoglucuronide in Gunn rat bile after injection of the pigment provides confirmatory evidence for the absence of a UDP-glucuronic acid-independent process.

Critique of the Assay and Significance of Bilirubin Conjugation

Publisher Summary This chapter describes the methodology and properties of uridine diphosphate (UDP)-glucuronyltransferase and of the related UDP-glycosyltransferase activities. It also delineates the applications to human disease. Bilirubin derives largely from senescent erythrocyte hemoglobin. In most normal adult animals, conjugation of bilirubin with various sugars represents the major mechanism for its transformation. The reaction products are eliminated from the organism by excretion into the bile. Glucuronyl transfer is of predominant importance in the biotransformation of bilirubin in man and rat. Glucuronyl transference to bilirubin and to many other acceptor substrates (G5, L14, M14, P4, W4), with the exception of phenolphthalein (G5), is higher in the rough endoplasmic reticulum than it is in the smooth endoplasmic reticulum. The main field of application of the in vitro assays of bilirubin UDP glycosyltransferase activities is the evaluation of stimulation or inhibition—by drugs or endogenous substances—of the enzyme activities or of their maturation. Development of unconjugated hyper-bilirubinemia in some neonates is related to breast feeding.

Purine and pyrimidine excretion: I. Chromatographic separation and identification of purine and pyrimidine metabolites in human urine

Abstract A number of purines and pyrimidines and other ultraviolet absorbing compounds can be demonstrated by column and paper chromatographic methods in the urine of normal persons and patients with liver disease, confirming observations reported by others. A study of the influence of diet and of administration of nicotinamide was found useful in the identification of unknown compounds. Increased amounts of several compounds were found in the urine of patients with liver disease.

Analysis of bilirubin conjugates

Publisher Summary The assays of enzymic formation of bilirubin’ monoglucuronides from bilirubin and bilirubin diglucuronide from bilirubin monoglucuronides are technically difficult because substrates and reaction products are labile and little or no adequate methodology was available in the past for the isolation and specific measurement of bilirubin conjugates. Contaminants present in substrates or formed during storage, incubation, and handling complicate the interpretation of the results of enzymic assays, and hence should be avoided. This chapter discusses measures for inhibiting artificial changes and the breakdown of rubins and describes simple techniques for structure elucidation, measurement, and the assessment of the purity of conjugated bilirubins. Bilirubin from commercial sources is usually employed as a substrate for the assay of UDPglucuronyltransferase activity and the synthesis of bilirubin glucuronides. As the composition of such bilirubin preparations may widely vary, it is mandatory to purify impure preparations by recrystallization.

Modelling of interaction of basic lipophilic ligands with cytochrome P-450 reconstituted in liposomes. Determination of membrane partition coefficients of S-( − )-nicotine and N,N-diethylaniline from spectral binding studies and fluorescence quenching

The spectral interaction of N,N-diethylaniline and S-(-)-nicotine with cytochrome P450IIB4 reconstituted into large unilamellar vesicles could properly be described by a model for interaction of basic lipid-soluble ligands with membrane-bound acceptor sites in which linear partitioning of non-ionized ligand in the membrane is postulated. Apparent spectral dissociation constants Ksapp for type I binding of N,N-diethylaniline and for type II binding of S-(-)-nicotine increased linearly with increasing lipid volume fraction alpha L of the proteoliposomes. From plots of Ksapp vs. alpha L, the membrane partition coefficient of each ligand was calculated. The apparent affinity of cytochrome P450IIB4 for the ligands increased as the pH was raised from 6.0 to 8.5. However, effective dissociation constants were virtually independent of the pH, indicating that only the uncharged form of the basic ligands interact with cytochrome P450IIB4. For each compound, the apparent quenching rate constants kqapp derived from the Stern-Volmer plots for dynamic quenching of the fluorescence intensity of 8-(2-anthryl)octanoic acid in liposomes, decreased with increasing liposomal concentration. Plots of (kqapp)-1 vs. alpha L of the liposomes yielded the overall bimolecular quenching rate constant kq of each quencher. The kq value for S-(-)-nicotine was about three orders of magnitude less than that for N,N-diethylaniline. The values of the partition coefficient of N,N-diethylaniline, obtained from the binding studies and the fluorescence quenching measurements, were identical (on average, Kp amounted to 383). Analysis of the quenching data of N,N-diethylaniline with Scatchard plots likewise revealed that the association of the compound with liposomal membranes is a pure partition process.

Modelling of chemical reactions catalysed by membrane-bound enzymes. Determination and significance of the kinetic constants

A model of multiphasic systems, based on the assumption of zero-order partition of substrates and products into the membranes, is applied to reversible mono-substrate and bi-substrate reactions catalysed by membrane-bound enzymes. Apart from replacement of single-phase kinetic constants by apparent kinetic constants, the derived kinetic expressions are formally identical with those for corresponding single-phase systems. The model confers to the apparent kinetic constants an experimentally verifiable meaning. For full characterization of membrane-kinetic systems, experiments at various concentrations of enzyme-embedding phospholipid are required. Extrapolation to zero phospholipid concentration of each Km app then yields the corresponding true kinetic constant characteristic of the membrane-bound enzyme and also provides a technique for determination of the membrane-partition constants. The procedure implies that the phospholipid content should be assayed for full characterization of membrane-bound enzymes. If, for practical reasons, the assays have to be limited to a single enzyme concentration, correction of the apparent kinetic constants is still possible provided the phospholipid concentration and the partition constants of the reactants are known. The model has permitted prediction of a number of previous observations reflecting the multiphasic nature of the systems. The assumptions, underlying the model, and their implications are examined as well as some commonly used experimental designs for determination of the type of enzymic site.

Comparative aspects of bile pigment formation and excretion

Breakdown of haem which is of key importance in most organisms, involves oxidative CO-evolving cleavage of the macrocyclic ring with formation of biliverdin-IX. In two major pathways established so far formation of biliverdin-IX alpha is followed by (a) biliary secretion or (b) reduction to bilirubin-IX alpha, formation of more hydrophilic derivatives (usually glycosidic conjugates) and biliary secretion. The scattered comparative information available indicates marked species variation with regard to the methin-bridge carbon atom removed from haem and the metabolic site of cleavage, the nature of bilirubin conjugates and the developmental sequence of maturation of enzyme activities and transport proteins involved in the chain of events leading from breakdown of haem to the excretion of the final end products.