The isolation and characterization of bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile.

The chemical structure of the major conjugate of bilirubin was unequivocally elucidated by structural analysis. The conjugated bilirubins were first separated from the lipid components of human duodenal aspirates or dog gall-bladder bile, and then resolved by t.l.c. into a series of tetrapyrroles. The major tetrapyrrole was then converted into its more stable dipyrrolic azo derivative for further analysis. The conjugated moiety of the azopigment was characterized after methanolysis with sodium methoxide. This reaction yields two types of product, those soluble in water and those soluble in organic solvents. The organic-soluble fraction was shown by t.l.c. and mass spectrometry to contain the methyl esters of the dipyrrolic azo derivatives of bilirubin. The water-soluble materials were analysed by enzymic procedures, t.l.c., n.m.r. spectrometry and combined g.l.c. and mass spectrometry. This analysis showed that the only water-soluble product resulting from the methanolysis was glucuronic acid. The structure was identical with that of pure standards, on both mass spectrometry and n.m.r. spectroscopy. No contaminating moieties were found. Quantitative measurement indicated that the glucuronic acid had been released in a 1:1 molar ratio with the resulting methyl esters of the dipyrrolic azo derivatives of bilirubin. This unequivocally establishes bilirubin diglucuronide as the major pigment present in bile. Past problems with identification of bilirubin diglucuronide were shown to originate from procedures which resulted in incomplete separation and isolation of the azopigments of the conjugated bilirubins, owing to contamination by biliary lipids.     

The preparation and characterization of bile pigments.

Brief reduction of bilirubin with dilute sodium amalgam was shown to give chromogens containing both vinyl and ethylidene beta-substituents. Acid-catalysed rearrangement of these chromogens with bilirubin or mesobilirubin and subsequent dehydrogenation gave a range of new violins containing unconjugated ethylidene and vinyl substitutents. Rearrangements between mesobilirubinogen, bilirubin and mesobilirubin gave dihydrobiliviolins, mesobiliviolins, biliverdins, dihydrobiliverdins and mesobiliverdins of the IIIalpha, IXalpha and XIIIalpha series. In this way 30 compounds were prepared, purified by t.l.c. as dimethyl esters, and characterized by n.m.r., mass and electronic spectroscopy, and by chemical interconversion and degradation.     

The oxidation products of crude mesobilirubinogen

Bile pigment esters were separated by ascending t.l.c. Apparently pure pigments, obtained by ferric chloride oxidation of crude mesobilirubinogen, derived from commercial bilirubin by reduction with sodium amalgam, were shown to be complex mixtures. Successive chromatography of their dimethyl esters on silica gel in methyl acetate-methyl propionate-dichloromethane-carbon tetrachloride (1:1:1:1, by vol.), ethyl methyl ketone-1,2-dichloroethane (1:2, v/v) and benzene-ethanol (100:3, v/v) revealed two major blue pigments (verdins), six major violet pigments (violins) and a red pigment (rhodin) together with numerous minor components. i-Urobilin dimethyl ester, prepared from mesobilirubinogen by dehydrogenation with aqueous iodine, was resolved into three major and at least four minor components on silica gel-kieselguhr (3:1, w/w) in benzene-ethanol (25:2, v/v). The chemical nature of these pigments was investigated by oxidation, by visible and u.v. spectroscopy, by mass spectrometry and by n.m.r. spectrometry. The evidence suggests unusual rearrangement of bilirubin during reduction leading to the formation of IIIalpha and XIIIalpha isomers. Isomeric forms of mesobiliviolin IXalpha and of i-urobilin IXalpha may also be formed.     

Analysis of bilirubin and bilirubin mono- and di-conjugates. Determination of their relative amounts in biological samples.

1. A novel method for determination of the relative amounts of unconjugated bilirubin and sugar mono- and di-conjugates of bilirubin in biological samples, including serum, is described and illustrated by its application to the analysis of bilinoids in rat bile. 2. The method is based on specific conversion of the carbohydrate conjugates of bilirubin into the corresponding mono- or di-methyl esters by base-catalysed transesterification in methanol. Under the selected reaction conditions, unconjugated biliru-in remains intact and no dipyrrole exchange in the bilinoids is detectable; transesterification of bilirubin mono- or di-glucuronide is virtually complete (approx. 99%), and sponification is negligible (less than 1%); recovery of the pigments is approx. 95%. 3. The reaction products bilirubin and its methyl esters are separated by t.l.c. and determined spectrophotometrically; the two isomeric bilirubin-IX alpha monomethyl esters are separated and therefore can be determined individually. 4. Reference bilirubin mono- and di-methyl esters have been synthesized and characterized, and the two isomers of bilirubin-IX alpha monomethyl ester and bilirubin dimethyl ester were obtained individually, in crystalline form. 5. With this new method, virtually all bilinoids (over 99%) in normal rat bile have been found to be conjugated, with diconjugates (71%) predominating. A significantly increased proportion of monoconjugates is present in bile collected from heterozygous Gunn rats or from normal rats that were refused with large amounts of bilirubin.     

Rapid transesterification of bilirubin glucuronides in methanol

The current studies present evidence that bilirubin conjugates derived from rat bile undergo rapid transesterification, $\text{in}$$\text{vitro}$, in solutions containing methanol. The conjugates of bilirubin and the methyl esters formed from them by exposure to methanol were isolated by thin layer chromatography. The isolates were chemically quantitated for their bilirubin and glucuronic acid composition. Characterization of the bilirubin methyl esters was performed by mass spectrometric analysis of the trimethylsilyl and phenylazo derivatives.     

Synthesis of biliverdin and bilirubin 1-O-acyl-beta-D-glucopyranuronic acids.

Biliverdin and bilirubin mono- and di-beta-glucuronides were prepared by nucleophilic substitution of the 1-O-mesyl derivative of alpha-ethoxyethyl-protected glucuronic acid (compound II) with the tetrabutylammonium salts of biliverdin and bilirubin. Removal of the acetal-protecting groups by mild acid treatment yielded biliverdin glucuronides, which were reduced to bilirubin glucuronides. Depending on reaction conditions the pure beta-anomers or mixtures highly enriched in the beta-anomers were obtained. The biliverdin and bilirubin glucuronides were identical with pigments derived from bile. They were characterized as the IX alpha isomers and the beta-anomers by alkaline hydrolysis, n.m.r. spectroscopy, hydrolysis with beta-glucuronidase and conversion into dipyrrolic azopigments. Model reactions of the 1-O-mesylate (II) with other nucleophiles also were performed, i.e. the acetate anion and various alcohols.     

Identification of ecdysone 22-long-chain fatty acyl esters in newly laid eggs of the cattle tick Boophilus microplus.

The five major apolar ecdysone esters present in newly laid eggs of the cattle tick Boophilus microplus have been purified by h.p.l.c. The quantities of the apolar esters present in the eggs were increased by administration of ecdysone to the mature females. G.c.-m.s. analysis, as their methyl esters, of the fatty acids released from the apolar ecdysone derivatives by alkali, coupled with positive-ion fast-atom-bombardment m.s. of the intact ecdysone esters, showed that the compounds consisted of a series of fatty acyl esters of ecdysone. The position of esterification of the ecdysone was established by p.m.r. spectroscopy. The combined data show that the novel apolar derivatives of ecdysone consist of the 22-palmitate, -palmitoleate, -stearate, -oleate, and -linoleate esters respectively. Confirmation was obtained by comparison with synthetic ecdysone 22-palmitate. The significance of the ecdysone fatty acyl esters as a possible source of free hormone during embryogenesis is discussed.     

Role of human skin in the photodecomposition of bilirubin

1. Human skin epithelium and human skin were found to absorb both free bilirubin and serum-bound bilirubin from an aqueous buffered medium. The serum-bound bilirubin thus absorbed was readily released when human skin epithelium or human skin were transferred to media containing no bilirubin. 2. The K(m) values for serum-bound bilirubin were 1.8x10(-3)m and 2.2x10(-3)m respectively for human skin epithelium and human skin; corresponding K(m) values for free bilirubin were 3.0x10(-4)m and 5x10(-4)m. The V(max.) for bound and free bilirubin was of the same magnitude, the apparent V(max.) being 1.0 and 1.66mumol/g of tissue for human skin epithelium and human skin respectively. 3. When human skin that had acquired a yellow tinge by absorbing bilirubin was incubated in a buffered medium and exposed to a mercury-vapour light, the yellow colour disappeared and decomposition products of bilirubin accumulated in the medium. 4. Experiments with [(3)H]bilirubin indicated that the pigment absorbed by skin was photo-oxidized to products that were soluble in water and the quantity and number of such products increased with the time of exposure of human skin to the light-source. Under similar conditions [(3)H]bilirubin alone in buffered medium was also oxidized and gave products which by paper chromatography appeared to be different from those released by human skin that had absorbed bilirubin. 5. The results suggest that by virtue of its large surface area human skin can act as a matrix for the degradative action of light on bilirubin.     

Preparation and properties of crystalline biliverdin IX alpha. Simple methods for preparing isomerically homogeneous biliverdin and [14C[biliverdin by using 2,3-dichloro-5,6-dicyanobenzoquinone.

Amorphous isomerically pure biliverdin IX alpha is readily prepared in more than 70% yield by dehydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide under carefully controlled conditions. Crystalline biliverdin IX alpha and amorphous [14C]biliverdin can be obtained similarly in more than 40+ yield. The pure crystalline pigment was characterized by elemental analysis, methylation, chemical and enzymic reduction to bilirubin, i.r.- and u.v.-visible-absorption spectroscopy, n.m.r. spectroscopy and field-desorption mass spectrometry, and its solubility was determined. Under certain conditions, dehydrogenation, gave biliverdin contaminated with III alpha and XIII alpha isomers as a result of disproporationation of bilirubin. Formation of non-IX alpha isomers depends on the concentrations of the reagents and the order in which they are mixed, and occurs under neutral anaerobic conditions. Free-radical reactions probably are responsible, suggesting that the first step in the deydrogenation of bilirubin with 2,3-dichloro-5,6-dicyanobenzoquinone in dimethyl sulphoxide is formation of a bilirubin cation radical, rather than hydride ion abstraction.     

Affinity labeling of the primary bilirubin binding site of human serum albumin.

A label for the bilirubin binding sites of human serum albumin was synthesized by reacting 2 mol of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) with 1 mol of bilirubin. This yielded a water-soluble derivative in which both carboxyl groups of bilirubin were converted to reactive enol esters. Covalent labeling was achieved by reacting the label with human serum albumin under nitrogen at pH 9.4 and 20 degrees. Under the same conditions, no covalent binding to the monomers of several proteins could be demonstrated. The number of binding sites for bilirubin and the label were found to be the same, and competition experiments with bilirubin showed inhibition of covalent labeling. The absorption, fluorescence and CD spectra of the label in a complex with human serum albumin were similar to those of the bilirubin human serum albumin complex. However, following covalent attachment to the spectral properties were changed, indicating loss of conformational freedom of the chromophore. Labeling ratios were selected to result in the incorporation of less than 1 mol of label/mol of human serum albumin. Under these conditions, labeling is thought to occur primarily at the high affinity binding site.     

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, Beer's 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.     

Bilirubin conjugates of human bile. Nuclear-magnetic-resonance, infrared and optical spectra of model compounds

N.m.r., i.r. and optical spectra of model compounds were recorded. These were to help in elucidating the structures of the phenylazo derivatives of bilirubin conjugates isolated from human bile. Model compounds included commercial and human bile bilirubin, mesobilirubin, bilirubin dimethyl ester, dimethoxybilirubin dimethyl ester and the corresponding phenylazo derivatives. The phenylazo derivative of vinylneoxanthobilirubinic acid was also investigated. All compounds were of the type IXα, and no other isomer could be detected with the spectroscopic methods employed. The compounds crystallize as the lactams, except for dimethoxybilirubin dimethyl ester and its phenylazo derivative, which are held in the lactim ether configuration. With all other compounds no tautomeric forms other than the lactams could be detected, although small proportions of bilirubin must exist as the lactim. Bilirubin does not form a betaine, a structure that has been proposed by von Dobeneck & Brunner (1965) to explain the bathochromic shift of its optical spectrum as compared with the expected position of the absorption maximum at 420nm. However, this shift to 453nm can be explained on the basis of internal hydrogen bonds occurring between the carboxylic protons and the pyrrole rings of bilirubin, as proposed by Fog & Jellum (1963), and new evidence for such a bonding has been accumulated. The bilirubin sulphate described by Watson (1958), which is formed by treatment of bilirubin with concentrated sulphuric acid and acetic anhydride, was also investigated. The main product of this reaction was isolated as its phenylazo derivative, and was shown to be 3,18-di(ethylidene sulphate)-2,7,13,17-tetramethylbiladiene-ac-8,12-dipropionic acid. The reaction leading to this compound is an addition of sulphuric acid to the vinyl side chains of bilirubin according to Markownikoff's rule.     

Uptake and release of bilirubin by skin

1. Skin epithelium of albino rat, mouse and guinea pig was shown to accumulate bilirubin from a medium containing free or bound bilirubin. 2. The K(m) values for bound bilirubin were 2.22x10(-3), 1.33x10(-3) and 9.5x10(-4)m for rat, mouse and guinea pig respectively and the corresponding K(m) values for free bilirubin were 5.2x10(-4), 4.0x10(-4), 1.8x10(-4)m; the V(max.) values of bound and free bilirubin were unchanged. 3. The uptake showed saturation kinetics. Bound bilirubin was released together with serum proteins. Free bilirubin bound to skin was not released into the medium. 4. Freezing and thawing of skin epithelium did not cause any significant lowering of the uptake of bilirubin but heat-denatured skin epidermis took up only 50% of the bound bilirubin or free bilirubin taken up by control unheated skin epithelium. 5. The uptake of free and bound bilirubin was prevented by HgCl(2) but not by sodium arsenate, NaCN, NaF, cycloheximide, 2,4-dinitrophenol or iodoacetate. 6. Most of the free bilirubin was bound to the lipids or lipoprotein fraction of skin epithelium and could be extracted by solvents. 7. Rat skin showed the highest accumulation and efflux of bilirubin.     

Methanolysis of cholesteryl esters: conditions for quantitative preparation of methyl esters.

We have investigated the conditions required to obtain a quantitative yield of methyl esters from cholesteryl esters by alkaline methanolysis. Methanolysis of the cholesteryl ester for 60 min at room temperature with 1 m methoxide reagent ensured complete reaction. Some ester hydrolysis always accompanied methanolysis and necessitated acid-catalyzed methylation of the resultant fatty acids after completion of the alcoholysis. Analysis of the composition of methyl ester product and remaining cholesteryl ester substrate before methanolysis had gone to completion showed selective hydrolysis of some fatty acid cholesteryl esters and illustrates the importance of obtaining a quantitative yield of methyl esters following methanolysis.     

Synthesis and conformational studies of 2-beta-chloro, 1-alpha-fluoro, and 2-beta-fluoro derivatives of 2-deoxy-N-acetyl-neuraminic acid

5-Acetamido-4,7,8,9-tetra-O-acetyl-2,3,5-trideoxy-2-fluoro-D-glycero-alp ha- and -beta-D- galacto -2- nonulosonic acid methyl esters and the beta-chloro analog were synthesized from N-acetylneuraminic acid. Their 1H- and 13C-n.m.r.spectra were completely assigned by using single-frequency decoupling, off-resonance decoupling, and spin-simulation programs. Bond angles estimated from the 1H coupling-constants indicate that all of the compounds adopt the 2C5 (L) conformation with minor conformational differences in the C3 side chain. 5-Acetamido-2,3,5,-tri-deoxy-2-fluoro-D-glycero-alpha- and -beta-D- galacto -2- nonulosonic acid and their methyl esters were also prepared.     

Studies of the conformation of bilirubin and its dimethyl ester in dimethyl sulphoxide solutions by nuclear magnetic resonance.

The conformation of bilirubin and its dimethyl ester in dimethyl sulphoxide (DMSO) was investigated by n.m.r. spectroscopy. The chemical shifts of the pyrrole NH and Lactam protons of bilirubin and its dimethyl ester in DMSO indicate a strong interaction with the solvent. Inter-proton distances were calculated from nuclear Overhauser effects (NOE), selective and non-selective relaxation times (T1) and rotational correlation times taken from 13C relaxation times. The interproton distances indicate that the conformation of the skeleton of bilirubin and its dimethyl ester in DMSO is similar to that of bilirubin and mesobilirubin in the crystalline state and in chloroform solutions, except for a possible slight twist of the pyrrolenone rings about the methine bonds, which may be a consequence of solvation of the NH groups by DMSO. Unlike in chloroform solutions, no direct hydrogen-bonding occurs between the carboxylic acid and the lactam groups of bilirubin in DMSO, as shown by the absence of an NOE between these groups. The fast exchange of the pyrrole NH protons with 2H shows that no hydrogen-bonding occurs between these protons and the propionic residues, in line with their solvation by DMSO. From the above results, and from the slowness of the internal motion of the propionic residues of bilirubin and its dimethyl ester, it is concluded that these residues are tied to the skeleton via bound solvent molecules.     

Studies on the mechanism of binding of serum albumins to immobilized cibacron blue F3G A.

The interaction of Cibacron Blue F3G A-Sepharose 4B with several serum albumins was studied. Although all albumins used were fond to bind to this adsorbent, human serum albumin was bound to a far greater extent than were the others. From the results of competition experiments and n.m.r. studies of Cibacron Blue and/or bilirubin binding to human serum albumin it is proposed that the mechanism of the interaction between human serum albumin and cibacron Blue is consistent wit Cibacron Blue binding to bilirubin-binding sites. In contrast with these findings with human serum albumin, there is little or no interaction of Cibacron Blue and the bilirubin-binding sites of albumins from rabbit, horse, bovine or sheep sera, although some interaction occurs between Cibacron Blue and the fatty acid-binding sites of these proteins. Structural analogues of Cibacron Blue have been used to investigate the binding of albumins to these ligands.     

Human hepatic beta-glucuronidase: an enzyme kinetic study

beta-Glucuronidase (EC 3.2.1.31) was purified from human liver and its activity was determined by enzyme kinetic method employing phenolphthalein glucuronic acid (PGA) and conjugated bilirubin, primarily bilirubin diglucuronide purified from human bile, as substrates in the absence or presence of D-glucaro-1,4-lactone. The enzyme was capable of acting on both PGA and conjugated bilirubin with Michaelis constants of 0.435 mmol/l at 56 degrees C and 1.02 mmol/l at 37 degrees C, respectively. Both reactions were beta-glucuronidase-specific because both were inhibited by D-glucaro-1,4-lactone in a competitive fashion. Conjugated bilirubin acted as a noncompetitive inhibitor of the enzyme for PGA in a two-substrate system. The study indicates that these two substrates bind at different catalytic sites of the enzyme and, on molar base, conjugated bilirubin had higher affinity for the enzyme and less degree of inhibition by D-glucaro-1,4-lactone than PGA. Whether such catalytic sites are also common for other beta-D-glucuronid ethers and esters remains to be proven.     

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.