Bilirubin conjugates of human bile. The excretion of bilirubin as the acyl glycosides of aldobiouronic acid,pseudoaldobiouronic acid and hexuronosylhexuronic acid,with a branched-chain hexuronic acid as one of the components of hexuronosylhexuronide

Structure elucidations have been performed on the bilirubin conjugates isolated from human hepatic bile as the phenylazo derivatives. The major bilirubin conjugates are excreted, not as was formerly thought in the form of glucuronides, but as the acyl glycosides of aldobiouronic acid, pseudoaldobiouronic acid and hexuronosylhexuronic acid. The isolated aldobiouronides are proposed to have the structures of an acyl 6-O-hexopyranosyluronic acid-hexopyranoside, an acyl 4-O-hexofuranosyluronic acid-d-glucopyranoside, and an acyl 4-O-β-d-glucofuranosyluronic acid-d-glucopyranoside respectively, with the acyl radicals being those of the phenylazo derivative of bilirubin. The pseudoaldobiouronide is suggested to be the acyl 4-O-α-d-glucofuranosyl-β-d -glucopyranosiduronic acid, with the acyl radical being that of the phenylazo derivative of vinylneoxanthobilirubinic acid. The hexuronosylhexuronide presumably is the acyl 4-O-(3-C-hydroxymethylribofuranosyluronic acid)-β-d-glucopyranosiduronic acid, with the acyl radical being that of the phenylazo derivative of bilirubin. The 3-C-hydroxymethylriburonic acid, isolated as one of the components of the hexuronosylhexuronide, is the first natural branched-chain hexuronic acid to be detected, and the first branched-chain sugar ever detected in humans.     

Biliary excretion of cyclohexylphenyl 4-[35S]sulphate in the guinea pig.

The metabolic fate and mode of excretion of cyclohexylphenyl 4-[35S]sulphate were studied in the guinea pig. Up to 54.8% of the dose appeared in the bile, the majority as unchanged ester. Substantial amounts of hydroxylated cyclohexylphenyl 4-[35S]sulphate were also excreted in the bile together with minor amounts of the corresponding glucuronic acid conjugate. When isolated guinea-pig livers were perfused with cyclohexylphenyl 4-[35S]sulphate the biliary components were the same as those in the intact animal, although the relative concentration of the hydroxylated derivative was significantly greater. When the hydroxylated derivative was re-injected into guinea pigs it was excreted almost entirely unchanged in the bile. However, in the rat, it was excreted in the bile as a glucuronic acid conjugate. These findings are discussed in relation to studies carried out in the rat [Hearse, Powell, Olavesen & Dodgson (1969) Biochem. Pharmacol. 18, 181--195] and to differences in enzyme activities in rat and guinea-pig liver. The results are also discussed in terms of the molecular-weight threshold for the excretion of anions in guinea-pig bile.     

The synthesis of esters of bilirubin

1-Alkyl-3-p-tolytriazenes were used to synthesize the methyl, ethyl, isopropyl and benzyl esters of bilirubin. Treatment of a chloroform solution of bilirubin with the triazene at room temperature gave high yields of the corresponding esters. These were identified by n.m.r. and mass spectroscopy together with elemental analysis. N.m.r. studies also suggest that bilirubin dimethyl ester is in the lactam rather than the lactim form.     

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, 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.     

Structure-Biological Activity Relationships in Alfalfa Antimycotic Saponins: The Relative Activity of Medicagenic Acid and Synthetic Derivatives Thereof Against Plant Pathogenic Fungi1

The antimycotic activity of medicagenic acid and of some synthetic derivatives thereof was tested against plant pathogenic fungi. In general they all possess antimycotic activity. Furthermore, in the case of Sclerotium rolfsii, compounds where the hydroxyl functions of the aglycon remained unchanged (medicagenic acid and its dimethyl ester) or could be enzymically released (3-0-β-D-glucoside of medicagenic acid dimethyl ester) were significantly more active than compounds where these functions were modified by acetylation or methylation. Selective 2-0-methylation of medicagenic acid and comparison of the antimycotic activity of the resulting derivative against S. rolfsii to that of other derivatives suggests that a potential free hydroxyl at position 3 is essential to antimycotic activity.     

Identification of ornithine and arginine conjugates of cholic acid by mass spectrometry.

Nalpha-Cholylornithine, -arginine, and -histidine were prepared according to a method previously employed for the chemical synthesis of the monoamino acid conjugates of bile acids. The products were shown to involve the alpha amino group of the dibasic amino acids by examination of the mass spectra of the original compounds, their lactams, their methyl esters and the methyl ester acetates. Only the methyl ester acetates gave detectable amounts of molecular ion. The free acids and the methyl esters of Nalpha-cholylornithine and -arginine gave identical lactams upon sublimation from the direct insertion probe. The synthetic Nalpha-cholylarginine was shown to yield a mass spectrum identical to that of an arginocholic acid recovered from the bile of an isolated perfused rat liver.     

Measurement of faecal bile acid sulphates

A method is described for the measurement, by difference, of the sulphate fractions of faecal bile acids. A solvolysis step (for the deliberate hydrolysis of the bile acid sulphates) was added to the procedure of sample homogenisation, extraction, enzymatic hydrolysis and thin-layer chromatography. The bile acids were quantitated by gas—liquid chromatography of their methyl ester and trifluoroacetate methyl ester derivatives on 3% QF-1 columns. The total bile acid excretion in 15 control subjects was 603 ± 71 mg/24 h (x̄ ± S.E.M.). The major bile acid peaks (mg/24 h) were: lithocholic acid, without solvolysis 118 ± 26 and including solvolysis 175 ± 30; deoxycholic acid 60 ± 8 and 90 ± 18 and chenodeoxycholic acid 13 ± 7 and 15 ± 7. It was concluded that bile acid sulphates may form a considerable proportion of the total bile acids excreted in man.     

Structure and enzymic activity of ribonuclease-A esterified at glutamic acid-49 and aspartic acid-53

The dimethyl ester of bovine pancreatic ribonuclease-A (dimethyl RNAase-A), the initial product of esterification of RNAase-A in anhydrous methanolic HCl, was isolated in a homogeneous form. The two carboxy functions esterified in this derivative are those of glutamic acid-49 and aspartic acid-53. There were no changes in the u.v.-absorption spectral characteristics, the accessibility of the methionine residues, the resistance of the protein to proteolysis by trypsin and the antigenic behaviour of RNAase-A as a result of the esterification of these two carboxy groups. Dimethyl RNAase-A exhibited only 65% of the specific activity of RNAase-A, but still had the same K_m value for both RNA and 2′:3′-cyclic CMP. However, the V_max. was decreased by about 35%. On careful hydrolysis of the methyl ester groups at pH9.5, dimethyl RNAase-A was converted back into RNAase-A. Limited proteolysis of dimethyl RNAase-A by subtilisin resulted in the formation of an active RNAase-S-type derivative, namely dimethyl RNAase-S, which was chromatographically distinct from dimethyl RNAase-A and had very nearly the same enzymic activity as dimethyl RNAase-A. Fractionation of dimethyl RNAase-S by trichloroacetic acid yielded dimethyl RNAase-S-protein and dimethyl RNAase-S-peptide, both of which were inactive by themselves but regenerated dimethyl RNAase-S when mixed together. Dimethyl RNAase-A-peptide was identical with RNAase-S-peptide. RNAase-S-protein could be generated from dimethyl RNAase-S-protein by careful hydrolysis of the methyl ester groups at pH9.5. The interaction of dimethyl RNAase-S-protein with RNAase-S-peptide appears to be about 4-fold weaker than that between the RNAase-S-protein and RNAase-S-peptide. Conceivably, the binding of the S-peptide `tail' of dimethyl RNAase-A with the remainder of the molecule is similarly weaker than that in RNAase-A, and this brings about subtle changes in the geometrical orientation of the active-site amino acid residues of these modified methyl ester derivatives. It is suggested that these changes could be responsible for the generation of the catalytically less-efficient RNAase-A and RNAase-S molecules (dimethyl RNAase-A and dimethyl RNAase-S respectively).     

Biliary excretion of foreign compounds. Benzene and its derivatives in the rat

1. The extent of the excretion in the bile of the rat of benzene and 21 of its simple derivatives was studied. 2. Some 16 compounds of molecular weight less than 200, and including neutral molecules (benzene and toluene), aromatic acids, aromatic amines and phenols, were injected in solution intraperitoneally into biliary-cannulated rats. Metabolites in the bile were identified and estimated. The extent of biliary excretion of these compounds was low, i.e. 0–10% of the dose in 24hr., and most appeared in the bile mainly as conjugates. 3. The biliary excretion of six conjugates of molecular weight less than 300, including three glycine conjugates, one sulphate conjugate, one glucuronic acid conjugate and two acetyl derivatives, was low (less than 3% of the dose). 4. It is concluded that simple benzene derivatives of molecular weight less than about 300 are poorly excreted in rat bile.     

Urobilinogen-i is a major derivative of bilirubin in bile of homozygous Gunn rats.

Gunn rats lack bilirubin UDP-glycosyltransferases, but diazo-negative derivatives of bilirubin have been described in their bile. In order to investigate this alternative disposal of bilirubin, crude bile samples from Gunn and Wistar rats were directly analysed by h.p.l.c. Besides bilirubin (in Gunn rats) or its glycosides (in Wistar rats), two major compounds were detected. A yellow one corresponded to the previously documented vitamin B-2 and was equally prominent in Gunn rats or Wistar-rat bile. The other compound was colourless, but on standing in contact with air it was spontaneously oxidized to a pinkish-yellow pigment. It was far more prominent in Gunn-rat bile. Analysis of bile obtained after intravenous injection of [14C]bilirubin to Gunn rats demonstrated that this compound was highly labelled. Freezing and thawing of the bile resulted in the formation of a series of diazo-negative derivatives, demonstrating that the original compound was quite labile. Spectral (adsorption and fluorescent) and chromatographic (h.p.l.c., t.l.c. and paper chromatography) analysis of the oxidized form of the labelled compound allowed its identification as urobilin-i. The colourless compound secreted in bile was urobilinogen-i. Administration of neomycin and bacitracin to Gunn rats or gut resection suppressed the biliary excretion of urobilinogen and thus confirmed its intestinal origin. Urobilinogen seems thus to represent the major bilirubin derivative present in Gunn-rat bile. Its breakdown products might represent the so-far-unidentified diazo-negative polar bilirubin derivatives. Since only a small amount of bilirubin is present in Gunn-rat bile, the urobilinogen formed in the intestinal lumen seems to be derived from bilirubin reaching the gut via routes other than the biliary one.     

Separation by thin-layer chromatography and structure elucidation of bilirubin conjugates isolated from dog bile.

1. A system for separation of bile pigments by t.l.c. and for their structure elucidation is presented. Separated bile pigments are characterized by t.l.c. of derived dipyrrolic azopigments. 2. At the tetrapyrrolic stage hydrolysis in strongly alkaline medium followed by t.l.c. demonstrates the presence of bilirubin-IIIalpha, -IXalpha and -XIIIalpha and allows assessment of their relative amounts. 3. Most structural information is derived from analysis of dipyrrolic azopigments. Such derivatives, obtained by treatment of separated bile pigments with diazotized ethyl anthranilate, were separated and purified by t.l.c. Micro methods showed (a) the nature of the dipyrrolic aglycone, (b) the nature of the bonds connecting aglycone to a conjugating group, (c) the ratio of vinyl/isovinyl isomers present in the aglycone and, (d) the nature of the conjugating groups (by suitable derivative formation and t.l.c. with reference to known compounds). 4. In bile of normal dogs at least 20 tetrapyrrolic, diazo-positive bile pigments could be recognized. Except for two pigments the tetrapyrrolic nucleus corresponded predominantly to bilirubin-IXalpha. All conjugated pigments had their conjugating groups connected in ester linkage to the tetrapyrrolic aglycone, Apart from bilirubin-IXalpha, monoconjugates and homogeneous and mixed diconjugates of bilirubin were demonstrated; conjugating groups of major importance were xylose, glucose and glucuronic acid. 5. Bilirubin isomer determination on native bile and isolated bile pigments, and dipyrrole-exchange assays with [14C8]bilirubin indicated (a) that the conjugates pre-exist in bile, and (b) that no significant dipyrrole exchange occurs during isolation of the pigments.     

13C and 1H NMR studies of helix-coil transition of poly(β-benzyl-L-aspartate) and poly(γ-benzyl-L-glutamate): Behavior in nonprotonating solvent mixtures,and origin of solvent-induced chemical shift

From the results of ¹³C-nmr measurement of poly(β-benzyl-L -aspartate) and its model compounds in dimethyl sulphoxide/deuterated chloroform mixtures, it was found that the side chain of poly(β-benzyl-L -aspartate) is solvated by dimethyl sulphoxide in the region more than dimethyl sulphoxide 20% (v/v), where the backbone maintains the α-helix. The chemical shift differences in the benzyl group carbons of poly(γ-benzyl-L -glutamate) (trifluoroacetic acid/deuterated chloroform) accompanied by the helix-coil transition, originate from the interaction between the ester group of the side chain and trifluoroacetic acid. The chemical shift difference in the ester carbon is similar. On the other hand, the chemical shift differences of the side-chain carbons in the alkyl portion (C^β, C^γ) originate not only from the interaction between the ester group of the side chain and trifluoroacetic acid, but also from some other unknown factors. The chemical shift differences of the side-chain carbons of poly(β-benzyl-L -aspartate) originate from the interaction between the ester group of the side chain and trifluoroacetic acid.     

Fluorescent derivatives of bile salts. II. Suitability of NBD-amino derivatives of bile salts for the study of biological transport.

Interaction of unconjugated and taurine-conjugated NBD-amino-dihydroxy-5 beta-cholan-24-oic acids bearing the fluorophor in the 3 alpha, 3 beta, 7 alpha, 7 beta, 12 alpha, or 12 beta position with albumin results in a small hypsochromic shift of the emission maximum and an increase in quantum yield, suggesting binding by hydrophobic interactions. The different unconjugated fluorescent bile salt derivatives are metabolized by intact rat liver in different ways. The unconjugated 3 beta-NBD-amino derivative is completely transformed to its taurine conjugate and secreted as such, whereas the 3 alpha-NBD-amino derivative is completely transformed to a polar fluorescent compound not identical with its taurine conjugate. The unconjugated 7 alpha- and 7 beta-NBD-amino derivatives are only partially conjugated with taurine and mainly secreted in unmetabolized form. The unconjugated 12 alpha- and 12 beta-NBD-amino derivatives are not at all transformed to their taurine conjugates, but are partially metabolized to unidentified compounds. They are predominantly secreted as the unmetabolized compounds. In contrast to the unconjugated derivatives, all taurine-conjugated fluorescent bile salt derivatives are secreted into bile unmetabolized. With the exception of the 3 alpha-compound, all synthesized taurine-conjugated fluorescent derivatives interfere with the secretion of cholyltaurine. Differential photoaffinity labeling studies using (7,7-azo-3 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2'-[2'-3H(N)]aminoethanesulfonate as a photolabile derivative revealed that in liver cells all fluorescent bile salt derivatives interact with the same polypeptides as the physiological bile salts. The hepatobiliary transport of taurine-conjugated NBD-amino bile salt derivatives is, due to hydrophobic interactions, accompanied by an increase in fluorescence intensity which is favorable for the study of biological bile salt transport by fluorescence microscopy.     

Bilirubin conjugates of human bile. Isolation of phenylazo derivatives of bile bilirubin

A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A(1), azo 2A(2) and azo 2A(3)) and azo 3A (azo 3A(1), azo 3A(2) and azo 3A(3)). The 2A pigments occur in approximately the following proportions: azo 2A(1), 90%; azo 2A(2), 10%; azo 2A(3), traces. The pigments are purified by crystallization, except for the A(3) pigments, which are probably degradation products arising from the corresponding A(2) pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol-di-isopropyl ether-ethyl acetate-methanol-0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B(1) to azo 2B(6) and azo 3B(1) to azo 3B(6) respectively) together with small amounts of products of hydrolysis (azo 2A(B) and azo 3A(B)). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B(2) type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B(1), 0-0.4%; azo 3B(2), traces; azo 3B(3), traces; azo 3B(4), 10%; azo 3B(5), 50%; azo 3B(6), 40%. Azo pigments 2B(1) to 2B(6) are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A(1) co-chromatographs with azo 3A(1), and azo 2B(6) co-chromatographs with azo 3B(6)), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).     

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.     

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.     

Synthesis of 3-(3-pyridyl)- and 3-(3-benzo[b]thienyl)-D-alanine

The DL-arylamino acid ethyl ester derivatives of beta-(3-pyridyl)-DL-alanine, and beta-(3-benzo[b]thienyl)-DL-alanine were synthesized by diethyl acetamidomalonate condensation with the respective arylmethyl halides followed by partial hydrolysis to the monoethyl ester and decarboxylation. Each derivative was enzymatically resolved to a separable mixture of the corresponding N-acetyl-L-amino acid and the unchanged D amino acid derivative. Acidic hydrolysis of the latter gave the corresponding D-amino acid, the optical purity of which was established by HPLC analysis of the 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC) derivative. The free D amino acids were converted to D-BOC derivatives by reaction with di-tert-butyldicarbonate in tert-butyl alcohol, water and sodium hydroxide.