Antony F. McDonagh

Phototherapy for Neonatal Jaundice

n engl j med 358;9 www.nejm.org february 28, 2008 920 A male infant weighing 3400 g was born at 37 weeks’ gestation after an uncomplicated pregnancy. The mother is a 24-year-old primipara who has type A Rh-positive blood. The infant’s course in the hospital nursery was uncomplicated. Although his mother needed considerable help in establishing effective breast-feeding, he was exclusively breast-fed. Jaundice was noted at the age of 34 hours. The total serum bilirubin level was 7.5 mg per deciliter (128 μmol per liter). The infant was discharged at the age of 40 hours and is seen in the pediatrician’s office 2 days later, now with marked jaundice. The results of his physical examination are otherwise normal, but his weight, at 3020 g, is 11% below his birth weight. His total serum bilirubin level is 19.5 mg per deciliter (333 μmol per liter), and his conjugated (direct) bilirubin level 0.6 mg per deciliter (10 μmol per liter). The complete blood count and peripheral-blood smear are normal. The infant has type A Rh-positive blood. The pediatrician consults a neonatologist regarding the need for phototherapy.

Crystallographic Analysis of Human Serum Albumin Complexed with 4Z,15E-Bilirubin-IXα

Bilirubin, an insoluble yellow-orange pigment derived from heme catabolism, accumulates to toxic levels in individuals with impaired or immature liver function. The resulting jaundice may be managed with phototherapy to isomerize the biosynthetic 4Z,15Z-bilirubin-IXα to more soluble and excretable isomers, such as 4Z,15E-bilirubin. Bilirubin and its configurational isomers are transported to the liver by human serum albumin (HSA) but their precise binding location(s) on the protein have yet to be determined. To investigate the molecular details of their interaction, we co-crystallised bilirubin with HSA. Strikingly, the crystal structure—determined to 2.42 Å resolution—revealed the 4Z,15E-bilirubin-IXα isomer bound to an L-shaped pocket in sub-domain IB. We also determined the co-crystal structure of HSA complexed with fusidic acid, an antibiotic that competitively displaces bilirubin from the protein, and showed that it binds to the same pocket. These results provide the first crystal structure of a natural bilirubin pigment bound to serum albumin, challenge some of the present conceptions about HSA–bilirubin interactions, and provide a sound structural framework for finally resolving the long-standing question of where 4Z,15Z-bilirubin-IXα binds to the protein.

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.

Commercial bilirubin: A trinity of isomers

Commercial preparations of the bile pigment, bilirubin, are used (frequently without further purifrcation) in metabolic investigations, as a convenient starting material for the preparation of other bile pig ments, as standards for clinical analyses, and in studies of the chemistry of bilirubin itself. The commercial material, which is obtained from bile or gallstones of bovine or porcine origin, is generally believed to consist essentially of bilirubin IX-a (fig. 1 B) formed metabolically via stereospecific cleavage of the a-methene bridge of protohaem-IX and the only isomer of bilirubin known to occur naturally. Although evidence has been published [l] suggesting that commercial bilirubin contains trace amounts of other isomers (bilirubin IX-0 and -6) arising from cleavage

The role of singlet oxygen in bilirubin photo-oxidation.

Summary Bilirubin photo-oxidation is a self-sensitised reaction involving singlet oxygen.

Turning green to gold.

Biliverdin is reduced to the potent antioxidant bilirubin in mammals and other vertebrates. The X-ray crystal structures of two biliverdin reductases, with different isomer specificities, show how substrate and cofactor bind to the enzymes and provide new insights into the mechanism of the reduction.

Molecular Mechanisms of Phototherapy of Neonatal Jaundice

The success of phototherapy depends on photochemical transformations of bilirubin within light-exposed tissues. These reactions alter the structure of bilirubin in such a way that the intact molecule or fragments of it can be excreted via the kidney or liver without having to undergo further metabolic modification. So far three photochemical reactions of bilirubin have been shown to occur in vivo, and it is likely that these three together account for most of the effect of light on bilirubin metabolism in jaundiced newborns. The three reactions are photooxidation, configurational isomerization, and structural isomerization. In this presentation I shall discuss briefly some of our evidence for the occurrence of these reactions in vivo and speculate on their relative contributions to the net effect of phototherapy. Since the interpretation of the in vivo studies is very much dependent on knowledge gleaned from in vitro photochemical studies, I shall begin with a discussion of the latter.

Origin of mammalian biliprotein and rearrangement of bilirubin glucuronides in vivo in the rat.

In hepatobiliary disease bilirubin becomes bound covalently to serum albumin, producing a nondissociable bile pigment-protein complex (biliprotein). To elucidate the mechanism of biliprotein formation we studied the bile pigment composition of blood from animals with experimental cholestasis and carried out comparative studies on the rate of biliprotein formation in vivo and in vitro during incubation of bilirubin glucuronides with albumin. Bile duct ligation in the rat and guinea pig led to rapid accumulation in the circulation of bilirubin, heterogeneous bilirubin esters of glucuronic acid, and a biliprotein that migrated along with albumin on high performance liquid chromatography. When the obstruction was removed, biliprotein remained longer in the circulation than did the other bile pigment species. Biliprotein and heterogeneous bilirubin esters of glucuronic acid were not formed in bile duct-ligated homozygous Gunn rats but they were formed when bilirubin glucuronides were incubated with Sprague-Dawley rat serum or human serum albumin at 37 degrees C in vitro. Bilirubin glucuronide rearrangement in vitro was accompanied by nonenzymic hydrolysis. We conclude that the formation of biliprotein in vivo is probably nonenzymic and suggest that mammalian biliprotein is formed by acyl migration of bilirubin from a bilirubin-glucuronic acid ester to a nucleophilic site on albumin.

Disposition and irreversible plasma protein binding of tolmetin in humans

The pharmacokinetics and irreversible plasma protein binding of tolmetin were studied in six healthy subjects after the administration of a single, 400 mg dose of tolmetin. With HPLC analysis, tolmetin, tolmetin glucuronide, and the isomers of tolmetin glucuronide, which result from intramolecular acyl migration in vivo, were detected in the plasma up to 4 hours after administration, whereas these conjugates were present in the urine up to 24 hours. Irreversible binding of tolmetin to plasma proteins occurred in all subjects. Irreversible binding exhibited a better correlation with exposure to tolmetin glucuronide (r = 0.5618) and the isomers of tolmetin glucuronide (r = 0.8200) than with exposure to tolmetin (−0.3635). This is consistent with the hypothesis that covalent binding occurs via the acyl glucuronide.

QUANTUM YIELDS FOR LASER PHOTOCYCLIZATION OF BILIRUBIN IN THE PRESENCE OF HUMAN SERUM ALBUMIN. DEPENDENCE OF QUANTUM YIELD ON EXCITATION WAVELENGTH

The quantum yield for laser photocyclization of bilirubin to lumirubin in the presence of human serum albumin (tHLR) was measured at five monochromatic excitation wavelengths in the range 450–530 nm. Solutions used were optically thin throughout the wavelength range and precautions were taken to exclude contributions from photocyclization of bilirubin XHIa impurities. The values obtained (7.2‐18 10‐4) were lower than those previously reported and showed the following wavelength dependence: 457.9 < 488.0 < 501.7 < 514.5 528.7. However, the rate of lumirubin formation, normalized to constant fluence, decreased with wavelength over the same wavelength range and no evidence was found that photoisomerization of bilirubin to lumirubin is faster with green (514.5 or 528.7 nm) than with blue (457.9 or 488.0 nm) light. The stereoselectivity of the configurational isomerization of bilirubin to 4Z,15E and 4E,15Z isomers also was studied. This reaction became less regioselective for the 4Z,15E isomer with increasing wavelength. The observed wavelength dependence of tHLR and of the [4Z,15E]: [4Z,15E] ratio at photoequilibrium are consistent with an exciton coupling model in which intramolecular energy transfer can occur between the two pyrromethenone chromophores of the bilirubin molecule in the excited state. Relative rates of lumirubin formation in vivo at different excitation wavelengths and constant fluence were estimated for different optical thicknesses and for different skin thicknesses. These estimates suggest that the recently reported clinical equivalence of blue and green phototherapy lights probably reflects the marked variation of skin transmittance with wavelength more than wavelength‐dependent photochemistry. The calculations also indicated that the optimal wavelength for phototherapy is probably on the long wavelength side of the bilirubin absorption maximum.