Bilirubin IXα Research Articles

A 1H-NMR study of bilirubin IXα solubilization by cholate micelles: application of nuclear overhauser effects

The solubilization of bilirubin IXα in aqueous solution by sodium cholate micelles has been examined by 270 MHz 1H-NMR spectroscopy. Incorporation of bilirubin into the micelles is accompanied by specific shifts of bilirubin vinyl and bridgehead protons and the C 18 and C 19 methyl groups of the steroid. The observed chemical shifts show a monotonic concentration dependence suggesting that changes in aggregation size are continuous. Nuclear Overhauser effects (NOE) have been shown to be a useful probe or micellization. A 4:1 cholate/bilirubin mixture has been investigated by difference NOE spectroscopy. The observation of intermolecular nuclear Overhauser effects between peripheral protons of bilirubin and cholate are diagnostic of spatially proximate groups. Inter-cholate nuclear Overhauser effects increase in magnitude upon bilirubin incorporation suggesting closer packing of steroid molecules on solubilization of the pigment. Intramolecular nuclear Overhauser effects observed for solubilized bilirubin are consistent with a compact intramolecularly hydrogen-bonded conformation resembling that determined for bilirubin in the solid state.

Solvent effects on the photoisomerization of bilirubin

Studies of the photolysis of bilirubin IX-α by absorbance difference (AD) spectroscopy show that in solvents which do not interfere with the intramolecular hydrogen bonding, e.g., CHCl3, EZ/ZE isomers and lumirubins are formed, with the former being predominant. The addition of ethanol to CHCl3, either before or during photolysis, greatly changes the AD spectra, possibly because of a reaction of photoproducts with the ethanol or solvatochromism of the EZ/ZE isomers. Solvents which interfere with the intramolecular hydrogen bonding of bilirubin, e.g., DMSO, increase the importance of the photoreaction that forms lumirubins. The kinetics of product formation indicate that the lumirubins result from a photoreaction of EZ/ZE isomers. The addition of aqueous buffer to DMSO lowers the extent of photoisomerization and leads to AD spectra that resemble those obtained for the photolysis in CHCl3. The addition of human serum albumin (HSA), using stopped flow techniques, to aqueous buffer solution of bilirubin immediately after photolysis gives evidence that the HSA traps the EZ/ZE isomers and prevents their reversion. However, in the presence of HSA the predominant pathway for photoreaction of bilirubin to EZ/ZE isomers is by reaction of the bound bilirubin. The AD spectra are similar to those obtained for solvents that interfere with hydrogen bonding.

Analysis of bilirubin conjugates in human bile by column liquid chromatography—Changes in their composition in hepatobiliary diseases

Bilirubin in human bile was analysed by high performance liquid chromatography (HPLC), and seven peaks were identifiable. There was no difference in the proportion of bilirubin between the B bile and C bile from healthy humans obtained by the Meltzer‐Lyon method. HPLC coupled with nuclear magnetic resonance spectroscopy on bilirubin after diazotization permitted differentiation between endovinyl and exovinyl conjugates of bilirubin.In patients with Gilbert's syndrome, type II Crigler‐Najjar syndrome, and haemolytic anaemia, the proportion of bilirubin diglucuronide (BDG) in the C bile decreased, and that of bilirubin monoglucuronide (BMG) increased. In patients with type II Crigler‐Najjar syndrome, marked elevations in BMG and bilirubin IXα and decreases in bilirubin monoglucuronide monoglucoside diester (BGG) and bilirubin monoglucuronide monoxyloside diester (BGX) were observed. In normal subjects and patients with unconjugated hyperbilirubinaemia, the diazobased indirect‐reacting bilirubin levels in blood and the proportion of BMG in bile were in good correlation. In patients with acute hepatitis, chronic hepatitis, and liver cirrhosis, there was no difference in the bilirubin composition in bile as compared to normal subjects, except for slight elevations in BGG observed in patients with chronic hepatitis and liver cirrhosis. In gallbladder bile obtained from patients with cholelithiasis, bilirubin IXα increased, suggesting some deconjugation of BDG.

Intramolecular Hydrogen Bonding in Bilirubin Bis-Tetra-n-butyl Ammonium Salts as Determined by1H-NMR Spectroscopy

Abstract Analysis of the N-H 1H-NMR chemical shifts of the bis-pro-pionate ions of bilirubin IXα and XIIIα reveals a preference in CDCl3 and d6-DMSO for folded conformations in which the propionate groups are intramolecularly H-bonded to the opposing pyrromethenone units.

The Diazo Reaction of Bilirubins and Phycorubins: A Quantitative Study*

A quantitative study of the diazo reaction has been made with several bilirubins including a 2,3- dihydrobilirubin and several phycorubins. The latter have been prepared from the integral phycocyanin of two cyanobacteria (Mastigocladus laminosus and Spirulina platensis), and from the phycocyanin subunits. These phycorubins have been subjected to the diazo reaction in order to test for the presence of a second covalent chromophore protein bond in the latter. 1)The diazo reaction of unsymmetrically substituted bilirubins, mesobilirubin (3) and bilirubin IXα (13) in aqueous solution yields four products, two isomeric 9-azo-dipyrromethenones, and two isomeric 9-hydroxymethyl-dipyrromethenones. The maximum total yield is ≥ 97% with diazotized sulfanilic acid, and ≤ 60% with diazotized ethylanthranilate. 2)The diazo reaction of the 2,3-dihydrobilirubin (16) yields likewise four products, two of them containing the 2,3-dihydrogenated ring A/B fragment. The attack is regioselective at C-11 (6:4). 3)The diazo reaction of the phycorubins with ethylanthranilate yields two peptide bound products, containing the ring A/B fragment, and two low molecular weight products. The latter correspond to the C/D fragment and are identical with the respective products derived from mesobilirubin 3. The attack is preferential at C-9 (≥ 4:1 with ≤ 1 mole reagent added). These results show, that there is no second covalent chromophore peptide bond in the PC investigated at the ring C/D fragment.

Enzymic formation of bilirubin IXα in a coupled oxidation system

Enzymic formation of bilirubin IXα in a coupled oxidation system

Presence of biliverdin IX.ALPHA. in the gallbladder bile of red sea bream.

The gallbladder bile of red sea bream Pagrus major was analyzed by TLC and HPLC.TLC pattern shows only one blue band and some yellow bands. Pigments extracted from blue band were analyzed by HPLC. Two peaks appeared. Judging from the absorbance at 365 and 436 nm and the retention time, the pigment in first minor peak may be bilirubin diglu-curonide. The retention time of second major peak was equal to that of biliverdin IXα. The blue pigment in second peak was reduced with NaBH4 and analyzed by HPLC. The retention time of reduced pigment was same as that of bilirubin IXα. The blue pigment was methylated with BF3-methanol and analyzed by HPLC. The retention times of methylated pigment and biliverdin IXα dimethylester were the same. These results indicate that the blue pigment is biliverdin IXα and only biliverdin IXα exists as verdinoids in the bile of red sea bream.

724—A spectroelectrochemical investigation of the oxidation of bilirubin IX-α in dimethylformamide

The electrochemistry of bilirubin IX-α in N,N-dimethylformamide has been investigated by cyclic voltammetry, modulated specular reflectance spectroscopy, small amplitude a.c. reflectance spectroscopy, OTTLE cell spectroelectrochemistry, and conventional spectroscopy. The data suggest a ECEC mechanism for oxidation of bilirubin to biliverdin. The oxidation peak potential for the mono-deprotonated bilirubin to its cation radical is +0.10 V ( versus 0.1 M Ag|Ag +). The reversible potential for the carbonium ion/free radical couple is −0.86 V. The peak potentials for biliverdin and purpurin oxidations are 0.24 and 0.47, respectively, while the reduction potentials for bilirubin and biliverdin are −1.35 and −1.55 V, respectively. Using the spectroelectrochemical techniques, it was possible to observe the UV-VIS absorption spectrum for each compound and several intermediates.

Bile pigments and bilirubin UDP-glucuronyl transferase during the metamorphosis of Rana catesbeiana tadpoles

1. 1. The major bile pigments in Rana catesbeiana tadpoles was bilirubin IXα. 2. 2. The concentration of bilirubin IXα increases in bile and plasma during metamorphosis. 3. 3. Bilirubin IXα and biliverdin IXα were also present in the bile of tadpoles. 4. 4. Bilirubin UDP-glucuronyl transferase activity was present in the livers of all tadpoles examined. 5. 5. Bilirubin UDP-glucuronyl transferase activity increases slightly during spontaneous metamorphosis and increases approximately 2-fold during T 3-induced metamorphosis.

The diazo reaction of bilirubin: structure of the yellow products: studies on plant bile pigments-14

The reaction of bilirubins with aromatic diazonium salts in alcoholic solvents leads to an equimolar mixture of two types of products. One is the well-known 9-azopyrromethenone. The other is a yellow product (λ max ≈ 420 nm) identified as 9-alkoxymethylpyrromethenone, the alkoxy-substituent being derived from the solvent. Thus, reaction of the symmetrically substituted bilirubins IIIα ( 1c) and XIIIα ( 1b) in methanol with diazotized sulfanilic acid yields one mole of the azopigments ( 4b and 4a), respectively, and one mole of the corresponding 9-methoxymethylpyrromethenones ( 2b and 2a). Bilirubin IXα ( 1a) consequently yields a mixture of all four products. The two resulting 9-methoxymethylpyrromethenones were separated by chromatography and identified as 2a and 2b. They can react further with the diazonium salts to give the corresponding 9-azopyrromethenones, but the reaction is much slower than that of bilirubin, which explains the observed product distribution. These results are discussed in relation to earlier work.

Pyrrole Exchange Reactions in the Bilirubin Series

AbstractAcid treatment of a mixture of bilirubin IXα (1) and octaethyl‐10,23‐dihydro‐21H,24H‐bilin‐1,19‐dione (7), followed by dehydrogenation and esterification gives biliverdin IXα, biliverdin XIIIα and biliverdin IIIα (as the dimethyl esters, 2, 3, 4, respectively), and octaethylbilindione (8), together with two minor verdins of intermediate polarity. These are identified as the products of the crossed pyrrole exchange reaction, which are 8‐carboxyethyl‐12,13,17,18‐tetraethyl‐2, 7‐dimethyl‐3‐vinyl‐21H,24H‐bilin‐1,19‐dione methyl ester (9) and 8‐carboxyethyl‐12,13,17,18‐tetra‐ethyl‐3,7‐dimethyl‐2‐vinyl‐21H,24H‐bilin‐1,19‐dione methyl ester (10). An improved preparation of bilirubin XIIIα (5) and bilirubin IIIα (6) is described and the products are crystallised and characterised. The NMR spectra of bilirubin XIIIα and IIIα are recorded in a polar aprotic solvent (DMSO‐d6) at 400 MHz, and certain unusual features are discussed.

A rapid and quantitative high performance liquid chromatographic method for assaying bilirubin and its conjugates in bile.

A rapid and quantitative high performance liquid chromatographic method for assaying bilirubin and its conjugates in bile has been developed. It is based on the use of ion-pair reverse-phase chromatography, utilizing heptanesulfonic acid in an acetate buffer, with a progressively increasing gradient of acetonitrile. The method resolves the following bilirubin IX alpha conjugates from bile: bilirubin diglucuronide, the two isomeric forms of bilirubin monoglucoside monoglucuronide diester, the two isomeric forms of bilirubin monoglucuronide, and bilirubin diglucoside. Unconjugated bilirubin is then eluted from the column by increasing the flow rate. The method also resolves the bilirubin XIII alpha and III alpha isomers of both bilirubin and the conjugates. In each case, the bilirubin XIII alpha precedes and the bilirubin III alpha follows the bilirubin IX alpha component. The high performance chromatographic run is completed in 35 min. The identity of the conjugates was ascertained by use of reference compounds isolated by thin-layer chromatography and by recollection of chromatographic peaks with identification of their diazo derivatives. The method was shown to have sufficient sensitivity that in vitro conjugating systems can be explored. Recollection and reinjection indicated that no isomeric scrambling occurs during the analytical procedure.

Chromatographic Separation and Spectroscopic Characterization of the Bilirubin Isomers IIIα, IXα, XIIIα and Their Dimethyl Esters

AbstractThe three structural isomers bilirubin IXα (1a), IIIα (2a), and XIIIα (3a) have been separated on a preparative scale by high‐performance low‐pressure liquid chromatography. They were converted into their dimethyl esters 1b, 2b, and 3b and characterized by UV, IR, MS, and fully assigned 1H NMR data. NMR is the only spectroscopic method which makes it possible to analyze mixtures of the three isomeric dimethyl esters quantitatively.

Jaundice in the Newborn

Pediatricians are confronted daily with the problem of the jaundiced newborn infant. The newborn infant is unique because (a) some elevation of serum bilirubin concentration is found in virtually all babies in the first few days of life, (b) the serum bilirubin concentration frequently rises to levels that are almost never encountered outside of the neonatal period, and (c) the newborn period remains the only time in which an elevated plasma bilirubin concentration per se represents a threat to the well-being of the organism. FETAL AND NEONATAL BILIRUBIN METABOLISM Chemical Structure and Properties of Bilirubin Bilirubin IXα (so called because it is derived from cleavage at the α position of the heme ring of ferropro-toporphyrin IX) is the end product of the catabolism of heme, of which the major source is circulating hemoglobin. Recent observations on the stereochemistry and conformation of the bilirubin molecule are helpful in understanding the potential neurotoxicity of bilirubin as well as the mechanism of phototherapy.1 The isomer, bilirubin IXα(Z,Z), is the major form of bilirubin as it exists in the blood where it is tightly bound to albumin. When this form of bilirubin takes up two hydrogen ions, it forms bilirubin IXα(Z,Z) acid, which is an involuted structure containing intramolecular hydrogen bonds.

Jaundice in the Newborn

Pediatricians are confronted daily with the problem of the jaundiced newborn infant. The newborn infant is unique because (a) some elevation of serum bilirubin concentration is found in virtually all babies in the first few days of life, (b) the serum bilirubin concentration frequently rises to levels that are almost never encountered outside of the neonatal period, and (c) the newborn period remains the only time in which an elevated plasma bilirubin concentration per se represents a threat to the well-being of the organism. FETAL AND NEONATAL BILIRUBIN METABOLISM Chemical Structure and Properties of Bilirubin Bilirubin IXα (so called because it is derived from cleavage at the α position of the heme ring of ferropro-toporphyrin IX) is the end product of the catabolism of heme, of which the major source is circulating hemoglobin. Recent observations on the stereochemistry and conformation of the bilirubin molecule are helpful in understanding the potential neurotoxicity of bilirubin as well as the mechanism of phototherapy.1 The isomer, bilirubin IXα(Z,Z), is the major form of bilirubin as it exists in the blood where it is tightly bound to albumin. When this form of bilirubin takes up two hydrogen ions, it forms bilirubin IXα(Z,Z) acid, which is an involuted structure containing intramolecular hydrogen bonds.

Degradation of hemin IX and synthetic hemins to α-bilirubins and their conjugates in the isolated perfused rat liver

Hemin IX was perfused through rat liver of a normal, untreated animal. Its degradation products, collected in the bile fluid over a period of 90 min, were found to consist of the bilirubin IX-α diglucuronide (56%), the mixture of bilirubin IX-α monoglucuronides (42%), and free bilirubin IX-α (2%). When the synthetic hemin XIII 2 was perfused with the same technique, it was found to be degraded in the same way. The bile fluid contained the diglucuronide of bilirubin XIII-α 10 (55%), the monoglucuronide of bilirubin XIII-α 9 (43%) and the free bilirubin XIII-α 8 (2%). Similar results were obtained when the iron 1,4-di(β-hydroxyethyl)-2,3,5,8-tetramethyl-6,7-di(β-carboxyethyl) porphyrin 3 was perfused; the diglucuronide of the α-bilirubin 11 comprised 65% of the excreted bile bilirubins, the monoglucuronide was 25% of the total and the free α-bilirubin 11 10% of the total. Perfusion of hematohemin gave 58% of the diglucuronide of α-hematobilirubin, as well as 40% of the monoglucuronides, and 2% of the free α-hematobilirubin. The simultaneous perfusion of hematohemin and of hemin IX produced an inhibition of the degradation of the hemin IX, while hematohemin was degraded as described above. It was concluded that the normal rat liver is prepared to dispose of exogenously added hemins by their oxidation to α-biliverdins, reduction of the latter to the corresponding α-bilirubin and excretion of their conjugated derivatives through the bile duct.

Kinetics of biliary excretion of the main two bilirubin photoproducts after injection into Gunn rats.

The kinetics of biliary excretion of the main two photoproducts after injection into Gunn rats were examined. The photoproducts that are obtained from experiments in vitro consist of unknown pigment, photobilirubin IXa and a small amount of (ZZ)-bilirubin IXa. It was confirmed previously that the first two photoproducts are identical with the main two photoproducts obtained in vivo. In experiments on four animals, the average of total biliary recoveries of unknown pigment was 81.4%, and that of photobilirubin IXa in the bile estimated by the Sigma-minus method was 29.8 min and that for unknown pigment was 4.3 min. The rate of thermal reversion of photobilirubin IXa to (ZZ)-bilirubin IXa in the bile at 37 degrees C was very rapid, i.e. its half-life was 6.2 min.

New Concepts in Phototherapy: Photoisomerization of Bilirubin IXα and Potential Toxic Effects of Light

New information is summarized, indicating that configurational photoisomerization of bilirubin at the 5 and 15 carbon bridges is the major mechanism of bilirubin photocatabolism in vivo, and that singlet oxygen photooxidation plays only a minor role. The literature is reviewed concerning potentially damaging photodynamic reactions that are observed in vitro with vitamins, proteins, lipids, and nucleic acids, and their possible relationship to the limited number of toxic side-effects that have been detected with clinical phototherapy of neonatal jaundice. Secondary toxic effects, mediated by bilirubin photoderivatives or by retina-neuroendocrine pathways are also considered. Areas requiring further investigation are delineated.

An accurate and sensitive analysis by high-pressure liquid chromatography of conjugated and unconjugated bilirubin IX-alpha in various biological fluids.

An accurate and sensitive method was developed for the complete separation of the native tetrapyrroles, such as bilirubin and its mono- and di-conjugates of glucuronic acid, glucose and xylose, by ion-pair reversed-phase high-pressure liquid chromatography. The application of this method was demonstrated by the analysis of bile pigments in human bile and urine, and the method also makes it possible to estimate very low UDP-glucuronyltransferase activity, such as is found in the human foetal and neonatal liver.

The separation of configurational isomers of bilirubin by high pressure liquid chromatography and the mechanism of jaundice phototherapy

When deoxygenated chloroform solution of bilirubin IXα (ZZ) was irradiated by blue light, ion-pair reversed-phase high pressure liquid chromatography technique revealed that the pigment was converted to a mixture containing IIIα, IXα, XIIIα (ZZ-configuration), and more polar geometric isomers (E-configuration). All azodipyrroles derived from each peak of Z- or E-configuration resulted in one of the exo- or endo-vinyl isomers, indicating that the bilirubin molecule is not affected by any of the phenomena except for geometric isomerization under this photochemical condition.