Calculated free bilirubin levels and neurotoxicity.

Although most bilirubin in the circulation is bound to albumin, a relatively small fraction remains unbound. The concentration of this 'free' bilirubin (B(F)) is believed to dictate the biologic effects of bilirubin in jaundiced newborns, including its neurotoxicity. The threshold at which B(F) produces changes in cellular function culminating in permanent cell injury and cell death has been the subject of considerable debate. The objective of this study was to compare calculated central nervous system (CNS) B(F) levels in Gunn rat pups during (i) peak postnatal hyperbilirubinemia and (ii) sulfadimethoxine-induced acute bilirubin encephalopathy (ABE) previously reported from our laboratory with those predicted in human neonates with peak total serum bilirubin (TSB) levels of 35 mg per 100 ml (599 micromol l(-1)), a clinical cohort that often evidence moderate-to-severe adverse post-icteric neurodevelopmental sequelae. Homozygous j/j Gunn rat pups with neonatal hyperbilirubinemia due to a deficiency of the bilirubin conjugating enzyme uridine-diphosphate-glucuronosyl transferase 1A1 were studied along with non-jaundiced littermate heterozygous J/j controls. Sulfadimethoxine was used to displace bilirubin from albumin in hyperbilirubinemic j/j Gunn rat pups to increase their brain bilirubin content and induce ABE. Calculated Gunn rat CNS B(F) levels were determined as a function of genotype, sulfadimethoxine exposure and albumin-bilirubin binding constant. These data were compared with the human CNS B(F) predicted from the calculated serum B(F) in human neonates with a TSB of 35 mg per 100 ml as a function of albumin-bilirubin binding constant, albumin concentration and the assumption that at this hazardous bilirubin level there may be rapid equilibration of B(F) between serum and brain. There was a large gap between the upper limit of the calculated CNS B(F) 95% confidence interval (CI) range in non-jaundiced J/j pups (for example, 112 nM at k=9.2 l micromol(-1)) and the lower limit seen in the saline-treated hyperbilirubinemic j/j pups (556 nM at k=9.2 l micromol(-1)) as well as between the upper limit in saline-treated hyperbilirubinemic j/j pups (1110 nM at k=9.2 l micromol(-1)) and the lower limit seen in sulfadimethoxine-treated jaundiced j/j littermates (3461 nM at k=9.2 l micromol(-1)). There was considerable overlap and remarkable similarity between the predicted human CNS B(F) values at a TSB of 35 mg per 100 ml for a range of reported human serum bilirubin-albumin binding constants and albumin concentrations, and those calculated for saline-treated hyperbilirubinemic j/j Gunn rat pups. This exercise yielded strikingly similar apparent calculated neurotoxic B(F) levels for Gunn rat pups and human neonates rather than orders of magnitude differences that might have been predicted at the outset and add to a growing literature aimed at defining clinically germane neurotoxic B(F) thresholds.Journal of Perinatology (2009) 29, S14-S19; doi:10.1038/jp.2008.218.

Biliary excretion of a stretched bilirubin in UGT1A1-deficient (Gunn) and Mrp2-deficient (TR-) rats.

The metabolism and biliary excretion of a stretched bilirubin analog with a p-xylyl group replacing the central CH2 hinge were investigated in normal rats, Gunn rats deficient in bilirubin conjugation, and TR- rats deficient in bilirubin glucuronide hepatobiliary transport. Unlike bilirubin, the analog was excreted rapidly in bile unchanged in all three rat strains after intravenous administration. In TR- rats biliary excretion of the analog was diminished, but still substantial, demonstrating that the ATP-binding cassette transporter Mrp2 is not required for its hepatic efflux. These effects are attributable to differences in the preferred conformations of bilirubin and the analog.

Sodium etiobilirubin-IVgamma-C10-sulfonate: a highly solvated bile pigment structure containing two different non-ridge-tile conformers in the unit cell.

The crystal structure of the title compound is the first example of a bilirubin existing in both extended and cyclic conformations and the first bile pigment structure showing two markedly different conformations in the unit cell. In contrast to previous rubin structures the dipyrrinone rings are twisted out of planarity in both conformers. Because of numerous hydrogen-bonding and ionic interactions a highly complex tetrameric structure is observed in which each extended conformer is held pincer-like by another.

Synthesis and metabolism of the first thia-bilirubin.

A symmetrical C(10)-thiabilirubin analogue, 8,12-bis(2-carboxyethyl)-2,3,17,18-tetraethyl-7,13-dimethyl-10-thia-(21H,23H,24H)-bilin-1,19-dione (1), was synthesized from 8-(2-carboxyethyl)-2,3-diethyl-7-methyl-10H-dipyrrin-1-one in one step by reaction with sulfur dichloride. The thia-rubin exhibited the expected IR, UV-vis, and NMR spectroscopic properties, which are rather similar to those of mesobilirubin-XIIIalpha. Like bilirubin and mesobilirubin, 1 adopts an intramolecularly hydrogen-bonded conformation, shaped like a ridge-tile but with a steeper pitch. The longer C-S bond lengths and smaller bond angles at C-S-C, as compared to C-CH(2)-C, lead to an interplanar angle between the two dipyrrinones of only 74 degrees -or considerably less than that of bilirubin (approximately 100 degrees). On normal- and reversed-phase chromatography, 1 is substantially less polar than bilirubin. Despite this conformational distortion, 1 is metabolized in normal rats to acyl glucuronides, which are secreted into bile. In mutant (Gunn) rats lacking bilirubin glucuronosyl transferase, 1 (like bilirubin) was not excreted in bile.

Phototherapy: from ancient Egypt to the new millennium.

Phototherapy with ultraviolet light was widely and successfully used in the past for treatment of a variety of diseases. Phototherapy with visible light alone has no benefit except in the therapy and prophylaxis of unconjugated hyperbilirubinemia. For this purpose, radiation in the region of approximately 480 to 500 nm is most effective and radiation above approximately 550 nm is useless. The principle effect of the treatment is not photodegradation of bilirubin, but conversion of the pigment to structural isomers that are more polar and more readily excreted than the normal, more toxic "dark" form of the pigment. This, coupled with some photooxidation of bilirubin, diminishes the overall pool of bilirubin in the body and lowers plasma levels. In the future, phototherapy may be supplanted by pharmacologic treatment, but in the near future, the most likely advance will be the introduction of novel forms of light production and delivery.

Structure and metabolism of natural and synthetic bilirubins.

The secondary structure of bilirubin, with a ridge-tile shape and six intramolecular hydrogen bonds, is more stable than any other conformation and perhaps the most important determinant of its solubility and properties in solution and its hepatic metabolism/excretion. Uncoupling the intramolecular hydrogen bonding, increasing the acidity of the propionic acid, or even increasing its length can decrease the pigment's hydrophobicity and permit its excretion intact across the liver into bile; less intuitively obvious, so can subtle modifications at C(10), the pivotal carbon in the ridge tile.

Photoinduced covalent binding of frusemide and frusemide glucuronide to human serum albumin.

AIMS: To study reaction of photoactivated frusemide (F) and F glucuronide (Fgnd metabolite) with human serum albumin in order to find a clue to clarify a mechanism of phototoxic blisters from high frusemide dosage. METHODS: F was exposed to light in the presence of human serum albumin (HSA). HSA treated with this method (TR-HSA) was characterized by fluorescence spectroscopic experiment, alkali treatment and reversible binding experiment. RESULTS: Less 4-hydroxyl-N-furfuryl-5-sulphamoylanthranilic acid (4HFSA, a photodegradation product of F) was formed in the presence of HSA than in the absence of HSA. A new fluorescence spectrum excited at 320 nm was observed for TR-HSA. Alkali treatment of TR-HSA released 4HFSA. Quenching of the fluorescence due to the lone tryptophan near the warfarin-binding site of HSA was observed in TR-HSA. The reversible binding of F or naproxen to the warfarin-binding site of TR-HSA was less than to that of native HSA. These results indicate the photoactivated F was covalently bound to the warfarin-binding site of HSA. The covalent binding of Fgnd, which is also reversibly bound to the warfarin-binding site of HSA, was also induced by exposure to sunlight. Fgnd was more photoactive than F, indicating that F could be activated by glucuronidation to become a more photoactive compound. CONCLUSIONS: The reactivity of photoactivated F and Fgnd to HSA and/or to other endogenous compounds may cause the phototoxic blisters that result at high F dosage.

Structural volume changes upon photoisomerization of the bilirubin-albumin complex: a laser-induced optoacoustic study.

The Z-->E photoisomerization of the 1:1 complexes of human serum albumin (HSA) and several bilirubins (BR-IX alpha, -III alpha, meso-IX alpha and a mixture of -IX alpha, -III alpha and XIII alpha) affords in every case an almost negligible structural volume change (delta VR approximately 0) within detection limits (i.e. less than 2-4 cm3/mol for this isomerization with very low quantum yield) as determined by laser-induced optoacoustic spectroscopy. Based on previous model studies of photoisomerizations in aqueous environment, this negligible small change is interpreted as indicating that the part of the molecule undergoing photoisomerization is not exposed to water but is located in a hydrophobic protein cavity that shields the molecule from the aqueous medium. The BR-protein interaction within this cavity seems to be very loose in view of the small structural volume change observed. The energy difference between the Z and E isomers of the BR-HSA complexes was estimated to be less than 4 kJ/mol, probably close to zero (delta H approximately 0).

Porphyria and porphyrinology--the past fifteen years.

The porphyrias are diseases caused by defective biosynthesis of heme. Leavened by digressions on porphyria trivia, this article presents selected highlights from the last 15 years of research on the chemistry, diagnosis, and treatment of the porphyrias. Thanks largely to genetic analysis and new light shed on the magical chemistry of heme biosynthesis, this period has seen great advances in the understanding of porphyria. Sequence analyses of the genes for all of the enzymes required for heme biosynthesis have revealed the porphyrias as highly heterogeneous, with multiple mutations underlying each type. As a result of technical advances, clinical porphyrin analyses are easier and more detailed, but their misapplication to "multiple chemical sensitivity syndrome" or "intoxication porphyria" is unfortunate. The prospect of gene therapy shines ever brighter but is neither safe nor effective enough to be considered for porphyria. As practical spin-offs, porphyrins are in use increasingly for diagnosis and treatment of cancer and as herbicides and pesticides. Accounts of alleged porphyria in "Prominent People" in the popular press continue to appear, generating fanciful misconceptions, often at the expense of patients with these fascinating diseases.

Aqueous dissociation constants of bile pigments and sparingly soluble carboxylic acids by 13C NMR in aqueous dimethyl sulfoxide: effects of hydrogen bonding.

pKas for the acid dissociation of the carboxyl groups of bilirubin in water have been reported recently to be 8.1-8.4, or higher. These high values were attributed to intramolecular hydrogen bonding. They have led to suggestions that monoanions of bilirubin predominate at physiologic pH and are the species transported most readily into hepatocytes by carriers. Such high aqueous pKas are inconsistent with recent 13C nuclear magnetic resonance (NMR) measurements on mesobilirubin XIII alpha, done on aqueous solutions containing dimethyl sulfoxide. To investigate whether the presence of dimethyl sulfoxide leads to unreliable values when using 13C NMR spectroscopy to determine pKas of carboxylic acids that can undergo intramolecular hydrogen bonding, we measured the pKas of 13C-labeled fumaric, maleic, and phthalic acids in solutions containing up to 27 vol% dimethyl sulfoxide. In addition, we used 13C NMR to estimate the pKas of 2,2'-methylenebis[5-carbomethoxy-4-methylpyrrole-3-[1-13C] propanoic acid], a model for the two central rings of bilirubin. Our results show that 13C NMR of aqueous dimethyl sulfoxide solutions can be used with confidence to measure pKas of intramolecularly hydrogen-bonded carboxylic acids. They support our previous estimates for the pKas of bilirubin and confirm that intramolecular hydrogen bonding has little effect on the acidity of bilirubins in water. Together with previous studies and chemical arguments they strongly suggest that reported aqueous pKas of > 8, or even > 6, for the carboxyl groups of bilirubin are incorrect and that arguments used to rationalize them are questionable.

Bile pigments as HIV-1 protease inhibitors and their effects on HIV-1 viral maturation and infectivity in vitro.

Using recently developed molecular-shape description algorithms, we searched the Available Chemical Directory for known compounds similar in shape to the potent HIV-1 protease inhibitor Merck L-700,417; 15 compounds most similar in shape to the inhibitor were selected for testing in vitro. Four of these inhibited the protease at 100 microM or less and the most active of the four were the naturally occurring pigments biliverdin and bilirubin. Biliverdin and bilirubin inhibited recombinant HIV-1 protease in vitro at pH 7.8 with K1 values of approx. 1 microM, and also inhibited HIV-2 and simian immunodeficiency virus proteases. The related pyrrolic pigments stercobilin, urobilin, biliverdin dimethyl ester and xanthobilirubic acid showed similar inhibitory activity at low micromolar concentrations. Biliverdin, bilirubin and xanthobilirubic acid did not inhibit viral polyprotein processing in cultured cells, but they reduced viral infectivity significantly. At 100 microM, xanthobilirubic acid affected viral assembly, resulting in a 50% decrease in the generation of infectious particles. In contrast, at the same concentrations biliverdin and bilirubin exerted little or no effect on viral assembly but blocked infection of HeLaT4 cells by 50%. These results suggest that bile pigments might be a new class of potential lead compounds for developing protease inhibitors and they raise the question of whether hyperbilirubinaemia can influence the course of HIV infection.

Current therapy for Crigler-Najjar syndrome type 1: report of a world registry.

This study represents a multicenter survey on the management of patients with Crigler-Najjar syndrome (CNS) type 1. The aim of the survey was to find guiding principles for physicians in the care of these patients. Fifty-seven patients were included. At the time of inclusion, 21 patients had received a liver transplant (37%). The average age at transplantation was 9.1 +/- 6.9 years (range, 1-23 years); the age of the patients who had not been transplanted at the time of inclusion was 6.9 +/- 6.0 years (range, 0-23 years). Brain damage had developed in 15 patients (26%). Five patients died, and 10 are alive with some degree of mental or physical handicap. In 2 patients, ages 22 and 23 years, early signs of bilirubin encephalopathy could be reversed, in 1 by prompt medical intervention followed by liver transplantation and in the other by prompt liver transplantation. Seven patients underwent transplantation with some degree of brain damage at the time of the surgery; 1 of these died after retransplantation, 2 improved neurologically, and 4 remained neurologically impaired. The age of 8 patients with and 13 without brain damage at or before transplantation was 14.3 +/- 5.9 and 5.9 +/- 5.4 years (P < .01), respectively. Therapy of CNS type 1 consists of phototherapy (12 h/d), followed by liver transplantation. Phototherapy, although initially very effective, is socially inconvenient and becomes less efficient in the older age group, thus also decreasing compliance. Currently, liver transplantation is the only effective therapy. This survey shows that, in a significant number of patients, liver transplantation is performed after some form of brain damage has already occurred. From this, one must conclude that liver transplantation should be performed at a young age, particularly in situations in which reliable administration of phototherapy cannot be guaranteed.

Dissociation constants of water-insoluble carboxylic acids by 13C-NMR. pK(a)s of mesobiliverdin-XIII alpha and mesobilirubin-XIII alpha.

High-field 13C-NMR of 13C-enriched compounds in dilute aqueous d6-Me2-SO solutions provides a simple, accurate method for measuring pK(a)s of sparingly soluble carboxylic acids. Using this method, we found the pK(a)s of mesobilirubin-XIII alpha to be 4.2 and 4.9, much lower values than reported recently for bilirubin, and of mesobiliverdin-XIII alpha to be 3.9 and 5.3.

On the acid dissociation constants of bilirubin and biliverdin. pKa values from 13C NMR spectroscopy.

Biliverdin and bilirubin are naturally-occurring tetrapyrrolic bile pigments containing two propionic acid side chains. These side chains, and their propensity for ionization, are critical in the biological disposition of the pigments. Surprisingly, accurate dissociation constants for the propionic acid groups of biliverdin are unknown, and a wide range of values, extending over some 4 orders of magnitude, has been suggested for the Ka values of the propionic acid groups of bilirubin in aqueous solutions. Recently, pKa values of 6.7-9.3 have been reported for bilirubin--values much greater than the value of approximately 5 typical of propionic acid groups. These curiously high values, currently being used to explain the biological transport and metabolism of bilirubin and related compounds, have been attributed to intramolecular hydrogen bonding. We have determined the pKa values of 99% 13C-enriched (13CO2H) [8(3),12(3)-13C2]mesobilirubin-XIII, alpha, the corresponding biliverdin, and several monopropionic model compounds by 13C NMR spectroscopy. This technique allows direct observation and quantitative measurement of the carboxylic acid and carboxylate anion carbon signals. Analysis of the variation of carboxyl 13C NMR chemical shift with pH gave rubin pKa values of 4.2 and 4.9 and verdin pKa values of 3.9 and 5.3 in aqueous buffers containing only a very small quantity (0.086 mol fraction) of dimethyl sulfoxide. When extrapolated to water, the pKa values are essentially unchanged. The data provide the first experimentally-determined pKa values for a biliverdin. They indicate that intramolecular hydrogen bonding has little effect on the acid dissociation of bilirubin and suggest that the equilibrium acidity of the bilirubin carboxylic acid groups is not abnormally high but similar to the thermodynamic acidity found in other carboxylic acids, as originally suggested by Overbeek et al. (Overbeek, J. T. G., Vink, C. L. J., and Deenstra, H. (1955) Recl. Trav. Chim. Pays-Bas 74, 81-84).

Hyperbilirubinemia results in reduced oxidative injury in neonatal Gunn rats exposed to hyperoxia.

Bilirubin is a potent antioxidant in vitro. To determine whether bilirubin also is an antioxidant in vivo, we studied markers of oxidative injury in the Gunn rat model exposed to hyperoxia. Homozygous jaundiced males were mated with heterozygous nonjaundiced females to obtain both jaundiced and nonjaundiced pups within a litter. Once delivered, the pups and their mother were placed in air (21% O2) or hyperoxia (> 95% O2) for 3 d. Both jaundiced and nonjaundiced pups were removed from the chambers daily. Animals were sacrificed and blood was drawn for determination of serum bilirubin, blood thiobarbituric acid-reactive substances (TBARS) by fluorescence assay, serum hydroperoxides, and serum protein oxidation. Tissues (liver, lung, and brain) were assayed for lipid peroxides (TBARS, conjugated dienes [CD], loss of polyunsaturated fatty acid content [PUFA]). We also measured a wide range of serum antioxidants including superoxide dismutase, catalase, glutathione, vitamins A, C, and E, and uric acid. Blood TBARS were significantly decreased in the jaundiced pups compared to the nonjaundiced pups on day 3 of hyperoxia, and blood TBARS were inversely correlated to serum bilirubin on day 3 of hyperoxia (R2 +/- .89). Similar decreases in serum lipid hydroperoxides and serum protein carbonyl content were detected in the jaundiced pups as compared to their nonjaundiced littermates. Other serum antioxidants were not increased in jaundiced animals compared to nonjaundiced animals. Relative lung weight was lower in jaundiced pups exposed to hyperoxia compared to similarly exposed nonjaundiced pups, suggesting a reduction in hyperoxia-induced lung edema. We detected no significant effects of bilirubin on parameters of lipid peroxidation in solid tissues. We conclude that serum bilirubin protects against serum oxidative damage in the first days of life in neonatal Gunn rats exposed to hyperoxia. We speculate that bilirubin is a functionally important transitional antioxidant in the circulation of human neonates and that it may be involved in modulation of injury due to hyperoxia.

Synthesis and mass-spectrometric characterization of human serum albumins modified by covalent binding of two non-steroidal anti-inflammatory drugs: tolmetin and zomepirac.

Human serum albumins modified by covalently bound tolmetin or zomepirac were synthesized as models for similar products formed in vivo from acyl glucuronides. Activated esters of both drugs were prepared with 1-ethyl-3-(3-dimethylaminopropyl)-carbodi-imide, and then allowed to react with human serum albumin. Tryptic digests of both protein products were analysed by HPLC to identify peptides containing covalently bound drugs, and binding sites on albumin were identified by high-performance tandem MS. Three binding sites were common to both products, i.e. lysine-195, -199 and -351. Three further modified residues were identified for the tolmetin-albumin product, i.e. aspartic acid 1, and lysine-524 and -536.

Stereoselective disposition of naproxen glucuronide in the rat.

The disposition of R- and S-naproxen glucuronides were investigated after intravenous administration (approximately 1.5 mg/kg) to normal male Sprague-Dawley rats and to rats pretreated with phenylmethylsulfonylfluoride, an inhibitor of esterases. The relative stability of the two glucuronides also was measured in vitro. Both diastereomers were hydrolyzed rapidly in vivo, liberating naproxen, but R-naproxen glucuronide was hydrolyzed faster than the corresponding S-diastereomer. This difference resulted in a larger plasma AUC(Nap):AUC(Nap-G) ratio for the R-glucuronide. There was, however, no marked difference in the apparent clearance of the R- and S-diastereomers. Administration of phenylmethylsulfonylfluoride had no significant effect on the disposition of the two diastereomers. In 0.15 M phosphate buffer (ph 7.4) at 37 degree C, the fastest degradation process for both diastereomers in vitro was acyl migration. Our results show that R-naproxen glucuronide is more labile than S-naproxen glucuronide in vivo and in vitro, and suggest that hydrolysis, rather than biliary excretion, is the major process leading to elimination of R-naproxen glucuronide in vivo in the rat. These results demonstrate that the rat may in certain situations be an inadequate model for studying the disposition of acyl glucuronides and that the metabolic disposition, and possibly toxicities, of diastereomeric metabolites of chiral drugs can be quite different even when the individual diastereomers have similar apparent clearances.

Stereoselective binding properties of naproxen glucuronide diastereomers to proteins.

The stability of naproxen glucuronide (NAP-G) diastereomers was investigated in buffer, 0.3% and 3% human serum albumin (HSA) solutions, and human plasma. R-NAP-G was found to be less stable in phosphate buffer than its S-diastereomer, whereas incubation media containing protein in general increased the degradation rate of NAP-G but also caused a change of the stereoselective stability where the R-NAP-G was more stable than S-NAP-G. Reversible binding of NAP-Gs to HSA (0.3%) was investigated and compared with the corresponding properties of naproxen (NAP) enantiomers. NAP-G diastereomers exhibited a considerable and stereoselective affinity to HSA, although less than that observed for the NAP enantiomers. In vitro irreversible binding of NAP-Gs to HSA, human and rat plasma proteins was also investigated. Irreversible binding was higher for R-NAP-G (50 microM) than for S-NAP-G (50 microM) in all incubation media. This stereoselective difference was observed with HSA containing medium as well as in rat and human plasma. Incubation with unconjugated NAP did not lead to irreversible binding. Preincubation of HSA with acetylsalicylic acid (approximately 11 mM) and glucuronic acid (50 mM) decreased the extent of irreversible binding suggesting involvement of lysine residues for covalent binding. Preincubation with S-NAP also decreased the irreversible binding yield.