Natural Bilirubin Research Articles

Conformational Control of the Photodynamics of a Bilirubin Dipyrrinone Subunit: Femtosecond Spectroscopy Combined with Nonadiabatic Simulations.

The photochemistry of bilirubin has been extensively studied due to its importance in the phototherapy of hyperbilirubinemia. In the present work, we investigated the ultrafast photodynamics of a bilirubin dipyrrinone subunit, vinylneoxanthobilirubic acid methyl ester. The photoisomerization and photocyclization reactions of its (E) and (Z) isomers were studied using femtosecond transient absorption spectroscopy and by multireference electronic structure theory, where the nonadiabatic dynamics was modeled with a Landau-Zener surface hopping technique. The following picture has emerged from the combined theoretical and experimental approach. Upon excitation, dipyrrinone undergoes a very fast vibrational relaxation, followed by an internal conversion on a picosecond time scale. The internal conversion leads either to photoisomerization or regeneration of the starting material. Further relaxation dynamics on the order of tens of picoseconds was observed in the ground state. The nonadiabatic simulations revealed a strong conformational control of the photodynamics. The ultrafast formation of a cyclic photochemical product from a less-populated conformer of the studied subunit was predicted by our calculations. We discuss the relevance of the present finding for the photochemistry of native bilirubin. The work has also pointed to the limits of semiclassical nonadiabatic simulations for simulating longer photochemical processes, probably due to the zero-point leakage issue.

In Vitro Evaluation of a Novel Synthetic Bilirubin Analog as an Antioxidant and Cytoprotective Agent for Pancreatic Islet Transplantation

Bilirubin is a natural cytoprotective agent and physiologic doses have proven to be beneficial in various models of organ and cellular transplantation. Recently, we showed that bilirubin has protective effects in models of pancreatic islet transplantation, preventing cell death associated with islet stress and suppressing the release of damage-associated molecular patterns. Despite these promising therapeutic attributes, the natural bilirubin used in these research studies is animal-derived (porcine), making it unsuitable for clinical application. In the current study, we synthesized two bilirubin analogs that can be produced without the use of animal-derived products. Antioxidant activity for the analogs was measured using the ferric-reducing-ability-of-plasma (FRAP) and 2,2V-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) assays. Dose-dependent cytotoxicity and cytoprotective effects were then demonstrated in isolated rat islets. Compound 1 showed similar antioxidant activity to natural bilirubin. Dose-dependent cytotoxicity was seen following treatment with Compound 1 and natural bilirubin at doses >40 μM, resulting in significantly increased cell death when compared to control islets (P < 0.05) or islets treated with doses ≤20 μM (P < 0.05). Following hypoxic challenge, islet cell death was reduced in islets treated with Compound 1 at 10 μM (17.27% ± 0.26%) compared to natural bilirubin at 10 μM (51.36% ± 0.71%; P < 0.0001) or 20 μM (59.02% ± 0.83%; P < 0.0001) and control islets (36.51% ± 0.44%; P < 0.0001). Compound 1 was found to have promising antioxidant and cytoprotective effects, limiting islet cell death in a model of islet transplantation hypoxic stress. Compound 1 may serve as a synthetic drug lead for clinical islet transplantation and further evaluation of this molecule and its analogs is warranted.

Bilirubin and its congeners: conformational analysis and chirality from metadynamics and related computational methods

The conformational properties of natural bilirubin, bisbutyricmesobilirubin-XIIIα, and their chiral derivatives (βS,β′S)-dimethylmesobilirubin-XIIIα and 8,12-(γR,γ′R)- dimethylbisbutyricmesobilirubin-XIIIα were investigated by metadynamics, an improved molecular dynamics computational methodology useful for conformational analysis. Applied to linear tetrapyrroles, which may be treated as two blade molecular propellers, our results are represented by conformational energy hypersurface maps built as functions of the two central dihedral torsion angles (φ1 and φ2) following rotations of two dipyrrinone units about the connecting CH2, and are compared successfully with maps previously obtained from molecular mechanics. With an emphasis on the conformationally more flexible 8,12-(γR,γ′R)-dimethylbisbutyricmesobilirubin-XIIIα, a conformationally mobile optically active bilirubinoid with butyric acid replacing propionic, metadynamics reveals global and nearby minima. Its electronic circular dichroism and UV–Vis spectra predicted by DFT calculations correlate well with the experimental spectra and are explained on the basis of equilibrium between the predicted conformational energy minima.

QM study of complexation between natural bilirubin and poly-terthiophene carboxylic acid–Mn(II) as a biosensor: Temperature and interferences e®ect

QM study of complexation between natural bilirubin and poly-terthiophene carboxylic acid–Mn(II) as a biosensor: Temperature and interferences e®ect

QM study of complexation between natural bilirubin and poly-terthiophene carboxylic acid–Mn(II) as a biosensor: Temperature and interferences effect

Bilirubin is an insoluble yellow pigment produced from heme catabolism and serves as a diagnostic marker of liver and blood disorders. Here, a systematic study of several interactions and arrangements between different forms of natural bilirubin and poly-5, 2[Formula: see text]-5[Formula: see text], 2[Formula: see text]-terthiophene-3-carboxylic acid/Mn(II)2complex, PTTCA–Mn(II)2, as a biosensor of bilirubin has been investigated extensively. The PTTCA–Mn(II)2biosensor detects natural bilirubin through the mediated electron transfer by the Mn[Formula: see text]. Initially, density functional theory (DFT) using B3LYP and different basis sets including 6-31G* and 6-311G** has been employed to calculate the details of electronic structure and electronic energies of natural biliverdin and [Formula: see text]-, [Formula: see text]- and [Formula: see text]-bilirubin. Next, the interaction of the PTTCA–Mn(II)2biosensor, being in three possible spin states, with [Formula: see text]-, [Formula: see text]- and [Formula: see text]-natural bilirubin with 1:1 and 1:2 stoichiometry using UB3LYP/6-31G* method has been investigated. Natural population analysis (NPA) calculations have been used to derive more suitable interaction sites of bilirubin with Mn[Formula: see text] ions in PTTCA–Mn(II)2biosensor. Investigation of different manganese complexes with bilirubin shows that the most stable complex is high spin state (total electron spin [Formula: see text]) rather than intermediate and low spin states with 1:2 stoichiometry. Also, the temperature effect and interferences from other biological compounds such as ascorbic acid, L-glutamic acid, uric acid, creatine, glucose and dopamine have been investigated. The nature of the interaction between manganese metal cations and natural bilirubin is also discussed employing NPA, molecular orbital (MO) analysis and Bader’s Atoms in Molecule (AIM) theory.

1348 Early Formation of Photoisomers During Phototherapy is Not Significantly Enhanced by Increasing Irradiance or Employing Photodiode Lights

BackgroundPhototherapy is the mainstain of treatment for neonatal jaundice. Photoisomers are more polar than native bilirubin IX-alpha (z, z) and can be excretedinbileandurine without conjugation. Rapid formation of PI may...

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.

In vitro production of bilirubin photoisomers by light irradiation using neoBLUE

The light-emitting diode is used as one of the new light sources for phototherapy. NeoBLUE (Atom Medical, Tokyo, Japan) incorporates blue light-emitting diodes for the treatment of neonatal hyperbilirubinemia. The authors compared the in vitro efficacy of neoBLUE with conventional phototherapy devices. The three light devices used included neoBLUE and two conventional phototherapy devices with six blue-white (BW) or six green (GR) fluorescent tubes. A bilirubin/human serum albumin solution (15 mg/dL) in 200 x 300 mm elliptical bag was irradiated with each three light device. The average light intensity of neoBLUE, BW and GR was 22.5, 10.2 and 2.6 microW/cm(2) per nm, respectively, for the irradiated area. Bilirubin photoisomers and native bilirubin were measured by high-performance liquid chromatography. In neoBLUE, BW and GR, the respective production rate of cyclobilirubin was 6.0, 3.7 and 3.9 x 10(-2) mg/dL/min, and the respective (4Z, 15E)-bilirubin/(4Z, 15Z)-bilirubin ratio after irradiation was 0.44, 0.33 and 0.12; the (4Z, 15Z)-bilirubin reduction rate at 20 min after irradiation was 60, 68 and 82%, respectively. The reduction rate of (4Z, 15Z)-bilirubin correlated with the (4Z, 15E)-bilirubin/(4Z, 15Z)-bilirubin ratio. Phototherapy using the neoBLUE under high level may be clinically more effective than therapy using the conventional light source from the results of the production rate of cyclobilirubin.

Modeling of biliverdin reduction process: regio-specificity and H-bonding

Octa ethyl biliverdin (OEBV) has been employed as a model for natural biliverdin and its geometry has been optimized by using semiempirical (AM1, PM3), DFT, and hybrid ONIOM methods. Geometries and energetics of formation of octa ethyl bilirubin (OEBR) formed by reduction from OEBV via three carbon sites β, γ, and δ have been obtained. It has been shown that γ-OEBR has two configurational isomers (named γ 1 and γ 2 ), which can convert to each other by internal 1,5-hydrogen shift. The results show that, within the accuracy level of semiempirical methods, all three isomers namely, β, γ 1 , and δ-OEBR are of similar stability whereas, at higher level of theory, γ 1 -OEBR is less stable than others. Moreover, γ 2 -isomer with one more of its pyrrole rings being aromatic can achieve a higher symmetry, and is the most stable among others by at least 5–6 kcal mol −1 based on various methods employed. It is interesting to note that the ridge-tile conformation, which has been confirmed for natural bilirubin was not observed for calculated geometries of γ 1 - and γ 2 -isomers. A conformational analysis show that an energy barrier of ∼25 kcal mol −1 must be surmounted for γ 2 to obtain the ridge-tile geometry. OEBV was synthesized and purified from octa ethyl porphyrin iron (III) chloride, and was reduced to OEBR by sodium borohydride (NaBH 4 ). Chemical reduction of OEBV with NaBH 4 was followed in CDCl 3 and CD 3 OD solutions and the product was characterized by 1 H NMR and UV–Vis spectroscopy. The results show that γ 2 -isomer as the major product, forms along with γ 1 via an equilibrium tautomerization reaction.

Aromatic congeners of bilirubin: synthesis, stereochemistry, glucuronidation and hepatic transport

A new synthetic analog ( 1) of the bile pigment bilirubin-IXα (bilirubin, Fig. 1) with phenyl groups replacing vinyl was prepared by a constitutional scrambling reaction of a mixture of two new, symmetric phenylrubin analogs ( 2 and 3) of bilirubin-XIIIα and IIIα. The former ( 2) with two endo-phenyls, and the latter ( 3) with two exo-phenyls were synthesized by condensation of a dipyrrylmethane dialdehyde with appropriate methylphenylpyrrolinones, which were prepared in several steps from 4-methyl-3-phenyl-2-( p-toluenesulfonyl)pyrrole, obtained by the Barton–Zard pyrrole synthesis. Nuclear Overhauser effect 1H NMR studies of 1– 3 confirm that, like their bilirubin equivalents, these yellow-orange pigments adopt an intramolecularly hydrogen-bonded ridge–tile conformation. Reverse phase HPLC and TLC suggest that 3 is less polar than 2, and that 1 has intermediate polarity. Large differences in the induced circular dichroism spectra of 1– 3 were found in pH 7.4 aqueous buffered solutions of human serum albumin. Despite the presence of bulky, lipophilic phenyl groups, 1– 3 are metabolized like natural bilirubin in rats and require glucuronidation by the same enzyme for canalicular secretion from the liver into bile. However, there are striking qualitative differences between the three pigments in the ratio of mono- to diglucuronides formed. Phenyl substituents at the exo positions of the lactam rings diminish the proportion of diglucuronide more than phenyl substituents at the endo positions.

Synthesis of a New Lipophilic Bilirubin. Conformation, Transhepatic Transport and Glucuronidation

Analogs of symmetrical bilirubin isomers with vinyl groups replaced by n-butyls ( 1 and 2) were synthesized and found to be much more soluble in nonpolar solvents than either bilirubin itself or its analogs with ethyls in place of vinyls ( 3 and 4). The increased lipophilicity of 1 and 2 allowed, for the first time, vapor pressure osmometric molecular wt. determinations of a bilirubin acid, showing in CHCl 3 solvent: MW obs=632±10 for 1, and 637±10 for 2 (both formula weight 644)—data that clearly indicate monomers and no dimers. The induced circular dichroism (ICD) spectra of 1 and 2, with negative exciton chirality Cotton effects, are quite like those of 3 and 4 in chloroform containing quinine, but the ICDs in aqueous buffer containing human serum albumin differ considerably. Rubins 1, 3 and 4 exhibit dissimilar but positive exciton chirality Cotton effects, while 2 shows a negative exciton chirality Cotton effect. The metabolism of the n-butyl rubins in the rat is highly dependent on the specific endo or exo location of the n-butyl groups. Rubin 2, with two endo n-butyl groups was metabolized rather like the corresponding mesobilirubin ( 4) or natural bilirubin itself, being converted to a mono and diglucuronide that were excreted promptly in bile. The presence of the bulky endo- n-butyl groups and the high lipophilicity of the compound seemed to interfere with glucuronidation in the liver only slightly. In contrast, the similar, yet even more lipophilic n-butyl rubin 1, which has two exo n-butyl groups, was glucuronidated and excreted in bile in the rat rather poorly.

138 BILIRUBIN PHOTOISOMERIZATION PRODUCTS IN URINE FROM A CRICLER-NAJAR TYPE 1 PATIENT

Crigler-Najjar type I syndrome (C.-N.s.) is a rare anomaly of bilirubin (BR) metabolism characterized by life-long hyperbilirubinemia. Phototherapy (PT) is the only treatment of value in the long-term management of C.-N.s.. In this study, we have analyzed the BR photoisomer composition in urine of a 18 year-old girl with C.-N.s., before, during and after PT administered with Philips F40T12/BB ‘special’ blue fluorescent lamps. The HPLC analysis of serum samples indicates that only native BR and its configurational isomer, 4Z,15E-BR, were present, with a relative steady-state concentration of about 24% of 4Z,15E-BR during PT. In urine, both configurational and structural [lumirubin, (15Z-LR + 15E-LK)] isomers were found before and, with increasing concentrations, during PT. Before PT, the total amount of structural isomer pool (15Z-LR+15E-LR) was as much as 3 times higher than configurational pool (4Z,15E-BR+4E, 15Z+BR+4Z,15Z-BR). This proportion did not change when the patient was exposed to PT light, although the total pigment concentration in urine increased markedly. This study suggests that the BR photo-product elimination in this adult with C.N.s. is significantly different than in the neonates. In fact, 15Z-LR (1-4%) and traces of 4E-15Z-BR are detected in serum of icteric babies exposed to PT, along with 4Z,15Z-BR and 4Z,15E-BR. On the other hand, the urine of icteric infants contains very small concentrations of the configurational pool. These evidences show that in C.-N.s. mechanisms exist to excrete all BR photoisomers. The existence of a relevant fraction of configurational photoisomers in urine suggests that their contribution to the mechanism of action of PT in C.-N.s. is not negligible.

Proton-induced dismutation of superoxide in aprotic media by bile pigments

Cyclic voltammetry of molecular oxygen in aprotic media (dimethylformamide) and in the presence of bilirubin and other bile pigments shows that superoxide anion (O2-.) undergoes proton-induced dismutation. Lactam hydrogens of bile pigments are sufficiently acid to induce (O2-.) disproportionation to O2 and H2O2. Because of its characteristic lipophilic behavior, a biological role for natural bilirubin similar to that of other non-enzymatic lipophilic scavengers of (O2-.) is suggested.

On the binding of bilirubin and its structural analogues to hepatic microsomal bilirubin UDPglucuronyltransferase.

Hepatic glucuronidation of the asymmetrical natural bilirubin molecule results in formation of two different positional isomers, bilirubin C-8 monoglucuronide and bilirubin C-12 monoglucuronide. In view of the existence of multiple isoforms of UDPglucuronyltransferase, which is the microsomal enzyme system responsible for bilirubin esterification, we performed kinetic analysis of microsomal glucuronidation of bilirubin and a number of its structural congeners to determine whether synthesis of the two monoglucuronide isomers involved two distinct substrate-binding sites or reflected two different modes of binding to a single catalytic site. Both isomers were found in all tested species (man, rat, guinea pig, sheep), but there were marked species differences in the C-8/C-12 ratio of monoglucuronide found in bile or formed by liver microsomes. Correspondence between in vivo and in vitro results for such regioselectivity of glucuronidation was excellent in each species. On the basis of our results of kinetic analysis of bilirubin esterification at variable pigment substrate concentrations and inhibition studies with alternative substrates, we postulate that both natural monoglucuronide isomers are synthesized at a single binding site. Possible mechanisms responsible for the markedly regioselective esterification of bilirubin by rat and sheep liver were investigated by study of glucuronidation of selected structural analogues of the pigment. Our results do not support explanations of regioselectivity of bilirubin glucuronidation in terms of (i) preferential binding of either the C-8- or C-12-containing dipyrrolic half of the asymmetrical bilirubin molecule or (ii) enantioselective complexation of bilirubin UDPglucuronyltransferase to one of the two chirality enantiomers of intramolecularly hydrogen-bonded bilirubin.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous esterification of bilirubin by liver microsomes. Evidence for an intramicrosomal pool of UDP-glucose and lumenal orientation of bilirubin UDP-glycosyltransferase.

Conjugation of natural bilirubin (BR) depends on a hepatic microsomal UDP-glycosyltransferase using UDP-Glc, UDP-xylose, and predominantly UDP-GlcA. We found that esterification of BR occurred when washed intact microsomes derived from rat or guinea pig liver were incubated with BR in the absence of added UDP-sugar. This endogenous esterification was shown to lead predominantly to formation of the two positional isomers of BR monoglucoside and displayed the same regioselectivity as found for the BR monoglucosides formed by microsomes incubated with a saturating concentration of added UDP-Glc. This finding and absence of endogenous esterification in liver microsomes from mutant rats lacking BR UDP-glycosyltransferase activities demonstrated that endogenous esterification depended on UDP-glycosyltransferase and indicated, therefore, that UDP-Glc was present in the intact microsomal vesicles. With UDP-Glc added to the extramicrosomal incubation medium, BR glucosidation was markedly enhanced when the membrane permeability barrier was disrupted by pretreatment of the microsomes with detergent, sonication, or Staphylococcus aureus alpha-toxin. In contrast, such membrane disruption resulted in abolishment of endogenous esterification of BR, and a direct relationship was found between impairment of endogenous esterification and degree of vesicle disruption, suggesting that the UDP-Glc on which endogenous esterification depended was present in the lumenal space of the microsomes. Kinetic evidence and absence of an effect of increasing the microsomal concentration of dolichol-P-Glc (Dol-P-Glc) on endogenous esterification excluded direct or indirect involvement of Dol-P-Glc in the endogenous esterification reaction. Preincubation of intact microsomes with UDP-Glc or UDP-xylose at 37 degrees C, but not at 0 degrees C, led to expansion of the microsomal UDP-sugar pool on which endogenous esterification depended, suggesting that both UDP-sugars can enter the microsomal vesicles by a temperature-dependent mechanism. In contrast to these findings, no increase of BR esterification was detected when the microsomes had been preincubated at 37 degrees C with UDP-GlcA. We conclude that native, intact microsomes contain a lumenal pool of endogenous UDP-Glc and that BR UDP-glucosyltransferase and UDP-xylosyltransferase, by virtue of a lumenal orientation, have direct access to the postulated intramicrosomal pool of nucleotide sugar.

Bilirubin Photoisomerization in Premature Neonates Under Low- and High-Dose Phototherapy

Photoisomerization of native bilirubin to more polar configurational isomers (Z,E-bilirubin) and structural isomers (lumirubin) was studied in 20 premature infants with physiologic jaundice to determine the effect of low-dose (6 microW/cm2/nm) v high-dose (12 microW/cm2/nm) phototherapy. Patients were assigned prospectively to receive either low- or high-dose treatment. Study groups were comparable with regard to birth weight, gestational age, and total bilirubin prior to the initiation of phototherapy. Treatment was administered with white light produced by a commercially available halogen-tungsten lamp. Dose was measured periodically during the study to ensure a uniform distribution of irradiance and constant exposure. Sera for photoisomers were obtained before initiation of treatment and at two, four, and eight hours. Photoisomers expressed as a percent of total bilirubin were determined using high-pressure liquid chromatography. Serum proportion of both configurational and structural isomers increased with the duration of phototherapy in both treatment groups. There was no significant difference between the percent of configurational isomers in low- and high-dose phototherapy groups. However, high-dose treatment produced a significantly higher proportion of the structural isomer lumirubin after four hours (0.7% low dose v 1.3% high dose, P less than .05). These data confirm that phototherapy results in both configurational and structural isomerization of bilirubin in vivo. Furthermore, the previously described "dose" effect of phototherapy may be attributed to the production of the structural isomer, lumirubin.

Executive Summary

A randomized, controlled study of phototherapy for neonatal hyperbilirubinemia was supported by the National Institute of Child Health and Human Development between 1974 to 1976. The study included 672 infants receiving phototherapy and 667 control infants. Phototherapy was effective in preventing hyperbilirubinemia in low-birth-weight infants (&amp;lt;2,000 g) who were placed under daylight fluorescent lights at 24 ± 12 hours of life for 96 hours. In this weight group, the number of exchange transfusions that were required due to hyperbilirubinemia was significantly lower in infants receiving phototherapy (4.1%) than in control infants (24.4%, P &amp;lt; .001). This was true even in infants with hemolysis and may be related to the fact that light treatment was initiated well before the development of hyperbilirubinemia. Phototherapy was effective in controlling hyperbilirubinemia in infants of birth weight 2,000 to 2,499 g when initiated after serum bilirubin concentration had risen above 10 mg/dL (mean 12.3 ± 1.9 mg/dL), as well as in infants with birth weight greater than 2,500 g whose serum bilirubin level had risen above 13 mg/dL (mean 15.7 ± 2.5 mg/dL) and in whom there was no hemolytic disorder. Light therapy instituted at these high levels of serum bilirubin did not effectively control hyperbilirubinemia in these two groups if hemolysis was present. Black infants responded to phototherapy as well as the nonblack infants. Phototherapy was most effective in the first 24 to 48 hours of its use. The pattern of declining efficacy after 48 hours is consistent with the thesis that unstable bilirubin isomers (photobilirubin) formed during the light treatment revert to natural bilirubin in the intestine following hepatic excretion and are reabsorbed via the enterohepatic circulation and contribute again to the bilirubin load to be cleared by the liver.

THE EFFECT OF SPECTRAL IRRADIANCE ON THE ISOMERIZATION OF BILIRUBIN IN VIVO

The initial event during phototherapy is the isomerization of native bilirubin to more polar configurational isomers (Z,E) and a structural isomer called lumirubin (LUM). The purpose of this study was to compare the effect of narrow spectrum blue light (B) versus broad spectrum white light (W) on the production of photoisomers in jaundiced infants. Thirteen preterm neonates (BW 1.68 ± .45 kg, GA 32 ± 2.3 wks) were studied under both B and W light. Irradiance at 450 nm was controlled at 12 uwatt/cm2/nm for both B and W. Infants were assigned to one of two study orders: B,W,B (n=4) or W,B,W (n=9). Each light condition (B or W) was administered for 12 hrs. Serum for Z,E and LUM were obtained before treatment and every 12 hours thereafter. Results were expressed as a percent of the total bilirubin. For all study infants % Z,E and LUM under B were compared with % under W for the two possible treatment sequences: W to B and B to W These data suggest that B is more effective than W in producing Z,E. The inability to demonstrate differences in % LUM may be due to more rapid excretion of this photoproduct.

The effect of retinoic acid on the surface charge distribution in cultured human chondrosarcoma and osteosarcoma cells : Y. Marikovsky, L. Meromsky∗ and R. Lotan Department of Membrane Research, The Weizmann Institute of Science, Rehovot 76100, Israel

From a concentrated solution of bilirubin in dimethylsulf oxide (about 13 mmol/1, standardized spectrophotometrically) and a serum diluted 1/3 (whose native bilirubin is lowered by exposure to phototherapy lamps) a bilirubin-enriched serum is prepared (bilirubin concentration about 130 μmol/1 = 7.5 mg/dl) wherein all the bilirubin is bound to albumin.Such an enriched serum behaves in the diazo-reaction in the same way as a bilirubin-enriched serum prepared with undiluted serum and can, therefore, be used as a working standard in serum bilirubin assays.

Theoretical constraints in the measurement of serum bilirubin binding capacity

The large majority of methods for measuring serum unoccupied bilirubin binding capacity involve adding an exogenous ligand to a diluted serum sample. If the added ligand binds specifically to bilirubin binding sites, the extrapolation is made that the amount of ligand which becomes bound represents the previously unoccupied bilirubin binding capacity of the original sample. This simple theory ignores the labile nature of the equilibrium reactions between bilirubin binding sites and the ligands with which they interact. The present theoretical and experimental study of these equilibrium reactions shows that (a) sample dilution alone results in changes in the proportional occupancy of bilirubin binding sites by bilirubin and other endogenous ligands, so that the extent of vacancy of those binding sites becomes exaggerated; (b) addition of an exogenous ligand to the diluted sample results is further displacement of native bilirubin and other endogenous ligands from bilirubin binding sites. The amount of added ligand which becomes bound is thus likely to represent a greater extent of vacancy of bilirubin binding sites than was present in the original sample. Results from such methods can therefore only overestimate the unoccupied bilirubin binding capacity of blood plasma in vivo. In order to avoid these analytical problems, current methods must be redesigned. It is suggested from this work that the ability of a serum to sequester safety additional bilirubin may best be assessed from measurement of its bilirubin buffering capacity. This parameter is different from the total or unoccupied binding capacity currently attempted, and its measurement is within the capability of published methods for measuring free (= unbound) bilirubin, modified to analyse minimally diluted samples.