Kinetics and specificity of feline leukemia virus subgroup C receptor (FLVCR) export function and its dependence on hemopexin.

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [(55)Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (K(d) < 1 pm) compared with albumin (K(d) = 5 nm) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7-25 mum) equivalent to that of the iron transport protein transferrin (22-31 mum), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.

Effective treatment of unconjugated hyperbilirubinemia with oral bile salts in Gunn rats.

BACKGROUND & AIMS: We tested the hypothesis that oral administration of bile salts, which are known to increase the biliary excretion of unconjugated bilirubin (UCB), decreases unconjugated hyperbilirubinemia in the Gunn rat model. METHODS: Adult Gunn rats were fed a standard diet or the same diet supplemented with 0.5 weight % ursodeoxycholic acid (UDCA) or cholic acid (CA) for 1 or 6 weeks. UCB and urobilinoids, a family of intestinal UCB breakdown products, were determined in plasma, feces, or both. After 6 weeks of treatment, tracer 3H-UCB was administered intravenously to determine steady-state UCB kinetics over the next 60 hours. RESULTS: One-week treatment with UDCA or CA decreased plasma UCB concentrations by 21% and 30%, respectively (each P < .01). During the first 4 days of treatment, both UDCA and CA increased the combined fecal excretion of UCB and urobilinoids (+52% and +32%, respectively; each P < .01). Prolongation of treatment to 6 weeks caused a persistent decrease in plasma UCB concentrations to approximately 40% below baseline (each bile salt P < .001). (3)H-UCB kinetic studies showed that UDCA and CA administration decreased UCB pool size (-33% and -32%, respectively; each P < .05) and increased UCB fractional turnover (+33% and +25%, respectively; each P < .05). CONCLUSIONS: Dietary bile salt administration induces a large, persistent decrease in plasma UCB concentrations in Gunn rats. Both UDCA and CA enhance UCB turnover by increasing its fecal disposal. These results support the application of oral bile salt treatment in patients with unconjugated hyperbilirubinemia.

Review: Bilirubin pKa studies: new models and theories indicate high pKa values in water, dimethylformamide and DMSO.

BACKGROUND: Correct aqueous pKa values of unconjugated bilirubin (UCB), a poorly-soluble, unstable substance, are essential for understanding its functions. Our prior solvent partition studies, of unlabeled and [14C] UCB, indicated pKa values above 8.0. These high values were attributed to effects of internal H-bonding in UCB. Many earlier and subsequent studies have reported lower pKa values, some even below 5.0, which are often used to describe the behavior of UCB. We here review 18 published studies that assessed aqueous pKa values of UCB, critically evaluating their methodologies in relation to essential preconditions for valid pKa measurements (short-duration experiments with purified UCB below saturation and accounting for self-association of UCB). RESULTS: These re-assessments identified major deficiencies that invalidate the results of all but our partition studies. New theoretical modeling of UCB titrations shows remarkable, unexpected effects of self-association, yielding falsely low pKa estimates, and provides some rationalization of the titration anomalies. The titration behavior reported for a soluble thioether conjugate of UCB at high aqueous concentrations is shown to be highly anomalous. Theoretical re-interpretations of data in DMSO and dimethylformamide show that those indirectly-derived aqueous pKa values are unacceptable, and indicate new, high average pKa values for UCB in non-aqueous media (>11 in DMSO and, probably, >10 in dimethylformamide). CONCLUSIONS: No reliable aqueous pKa values of UCB are available for comparison with our partition-derived results. A companion paper shows that only the high pKa values can explain the pH-dependence of UCB binding to phospholipids, cyclodextrins, and alkyl-glycoside and bile salt micelles.

Interactions of unconjugated bilirubin with vesicles, cyclodextrins and micelles: new modeling and the role of high pKa values.

BACKGROUND: Unconjugated bilirubin (UCB) is an unstable substance with very low aqueous solubility. Its aqueous pKa values affect many of its interactions, particularly their pH-dependence. A companion paper shows that only our prior solvent partition studies, leading to pKa values of 8.12 and 8.44, met all essential requirements for valid pKa determinations. Other published values, generally lower, some below 5.0, were shown to be invalid. The present work was designed to derive suitable models for interpreting published data on the pH-dependent binding of UCB with four agents, mentioned below, chosen because they are not, themselves, sensitive to changes in the pH range 4-10, and the data, mainly spectrometric, were of reasonable quality. RESULTS: These analyses indicated that the high pKa values, dianion dimerization constant and solubilities of UCB at various pH values, derived from our partition studies, along with literature-derived pH- and time-dependent supersaturation effects, were essential for constructing useful models that showed good qualitative, and sometimes quantitative, fits with the data. In contrast, published pKa values below 5.0 were highly incompatible with the data for all systems considered. The primary species of bound UCB in our models were: undissociated diacid for phosphatidylcholine, dianion for dodecyl maltoside micelles and cyclodextrins, and both monoanions and dianion for sodium taurocholate. The resulting binding versus pH profiles differed strikingly from each other. CONCLUSIONS: The insights derived from these analyses should be helpful to explore and interpret UCB binding to more complex, pH-sensitive, physiological moieties, such as proteins or membranes, in order to understand its functions.

The cytotoxic effect of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells is modulated by the expression level of MRP1 but not MDR1.

In vitro and in vivo studies have demonstrated that UCB (unconjugated bilirubin) is neurotoxic. Although previous studies suggested that both MRP1 (multidrug resistance-associated protein 1) and MDR1 (multidrug resistance protein 1) may protect cells against accumulation of UCB, direct comparison of their role in UCB transport was never performed. To this end, we used an inducible siRNA (small interfering RNA) expression system to silence the expression of MRP1 and MDR1 in human neuroblastoma SH-SY5Y cells. The effects of in vitro exposure to clinically-relevant levels of unbound UCB were compared between unsilenced (control) cells and cells with similar reductions in the expression of MRP1 or MDR1, documented by RT-PCR (reverse transcription-PCR) (mRNA), immunoblotting (protein), and for MDR1, the enhanced net uptake of a specific fluorescent substrate. Cytotoxicity was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] test. MRP1-deficient cells accumulated significantly more UCB and suffered greater cytotoxicity than controls. By contrast, MDR1-deficient cells exhibited UCB uptake and cytotoxicity comparable with controls. At intermediate levels of silencing, the increased susceptibility to UCB toxicity closely correlated with the decrease in the expression of MRP1, but not of MDR1. These data support the concept that limitation of cellular UCB accumulation, due to UCB export mediated by MRP1, but not MDR1, plays an important role in preventing bilirubin encephalopathy in the newborn.

Bilirubin inhibits the TNFalpha-related induction of three endothelial adhesion molecules.

Since an increased serum unconjugated bilirubin (UCB) level has been proposed as an independent protective factor against atherosclerotic disease, we investigated the molecular events at the basis of this effect. HUVEC and H5V cells were treated with TNFalpha and UCB and the effects assessed on E-selectin, VCAM-1 and ICAM-1. In HUVEC cells, UCB blunted the TNFalpha-induced gene upregulation of E-selectin VCAM-1 and ICAM-1. The same pattern was observed in H5V cells except for ICAM-1. UCB also inhibited the PMN endothelial adhesion in HUVEC H5V cells. Western blot and FACS analysis confirmed that UCB prevented TNFalpha-induced over-expression of adhesion molecules proteins in H5V cells. These data contribute to further explain the protective effect of bilirubin against development of atherosclerosis.

Unbound (free) bilirubin: improving the paradigm for evaluating neonatal jaundice.

BACKGROUND: The serum or plasma total bilirubin concentration (B(T)) has long been the standard clinical laboratory test for evaluating neonatal jaundice, despite studies showing that B(T) correlates poorly with acute bilirubin encephalopathy (ABE) and its sequelae including death, classical kernicterus, or bilirubin-induced neurological dysfunction (BIND). The poor correlation between B(T) and ABE is commonly attributed to the confounding effects of comorbidities such as hemolytic diseases, prematurity, asphyxia, or infection. Mounting evidence suggests, however, that B(T) inherently performs poorly because it is the plasma non-protein-bound (unbound or free) bilirubin concentration (B(f)), rather than B(T), that is more closely associated with central nervous system bilirubin concentrations and therefore ABE and its sequelae. CONTENT: This article reviews (a) the complex relationship between serum or plasma bilirubin measurements and ABE, (b) the history underlying the limited use of B(f) in the clinical setting, (c) the peroxidase method for measuring B(f) and technical and other issues involved in adapting the measurement to routine clinical use, (d) clinical experience using B(f) in the management of newborn jaundice, and (e) the value of B(f) measurements in research investigating bilirubin pathochemistry. SUMMARY: Increasing evidence from clinical studies, clinical experience, and basic research investigating bilirubin neurotoxicity supports efforts to incorporate B(f) expeditiously into the routine evaluation of newborn jaundice.

Highly sensitive method for quantitative determination of bilirubin in biological fluids and tissues.

Unconjugated bilirubin (UCB) exhibits potent antioxidant and cytoprotective properties, but causes apoptosis and cytotoxicity at pathologically elevated concentrations. Accurate measurement of UCB concentrations in cells, fluids and tissues is needed to evaluate its role in redox regulation, prevention of atherosclerotic and malignant diseases, and bilirubin encephalopathy. In the present study, we developed and validated a highly sensitive method for tissue UCB determinations. UCB was extracted from rat organs with chloroform/methanol/hexane at pH 6.2 and then partitioned into a minute volume of alkaline buffer that was subjected to HPLC using an octyl reverse phase (RP) column. Addition of mesobilirubin as an internal standard corrected for losses of UCB during extraction. Recoveries averaged 75+/-5%. The detection limit was 10pmol UCB/g wet tissue. Variance was +/-2.5%. When used to measure UCB concentrations in tissues of jaundiced Gunn rats, this procedure yielded UCB levels directly comparable to published methods, and accurately determined very low tissue bilirubin concentrations (

Bilirubin-induced cell toxicity involves PTEN activation through an APE1/Ref-1-dependent pathway.

Unconjugated bilirubin (UCB) is the major degradation product of the heme catabolism. A growing body of evidences suggests that UCB plays major biological effects by inhibiting cell proliferation in cancer cell lines and eliciting cell toxicity particularly in neurons and glial cells. Early molecular events responsible for bilirubin-induced cytotoxicity remain poorly understood. Using HeLa cells and mouse embryonic fibroblasts, we found that UCB at a concentration of free pigment (Bf) of 80 nM induced oxidative stress, promoting a significant increase in intracellular reactive oxygen species (ROS) and a decreased cell survival (by the MTT test). The ROS increase activated the antioxidant cell response through APE1/Ref-1, a master redox regulator in eukaryotic cells. Activation of APE1/Ref-1 was followed by a concomitant activation of Egr-1 transcription factor and by an upregulation of PTEN tumor suppressor, an Egr-1 target gene, leading to inhibition of cell growth. Blocking ROS generation with N-acetylcysteine pretreatment, restored cell survival, limited the upregulation of PTEN in response to UCB, and prevented the inhibition of cell proliferation. HeLa cells transfected with mutants of the PTEN promoter or silenced with APE1/Ref-1 small interference RNA confirmed that UCB modulates a signaling pathway involving APE1/Ref-1, Egr-1, and PTEN. These findings describe a new molecular pathway involved in the cytotoxic effects of UCB.

Revalidation and rationale for high pKa values of unconjugated bilirubin.

BACKGROUND: Our prior solvent partition analysis, published in 1992, yielded pKa values for unconjugated bilirubin of about 8.1 and 8.4, but these results have been challenged and studies by other methods have suggested pKa values below 5.0. METHODS: We repeated our published solvent partition studies, using 14C-unconjugated bilirubin highly purified by extraction of residual labeled impurities from CHCl3 into an aqueous buffer, pH 7.0. Partition ratios at six pH values from 5.0 to 9.0 were determined by radioassay and compared with our prior values obtained by diazo assay. RESULTS: At pH values ranging from 4.8 to 9.2, stable aqueous/chloroform 14C-partition ratios did not differ significantly from our published partition ratios based on diazo assay. CONCLUSION: These results support the high pKa values of unconjugated bilirubin, above 8.0, derived from our earlier solvent partition study. In both studies, our measurements were based on the rapid analysis of clearly under-saturated solutions of highly-purified bilirubin over a wide pH range, using properly purified and preserved solvents. No previous direct estimate of the aqueous pKa values of unconjugated bilirubin meets all these preconditions. Three theoretical factors acting in combination, each related to the unique, extensive internal H-bonding of the -COOH groups, are proposed to support high pKa values of unconjugated bilirubin in water: a) donation of an H-bond from the -OH moiety of the -COOH group, which is broken on ionization; b) hindered solvation of the -COO- group after ionization; and c) restricted rotation of the -COO- and -COOH groups. Our findings and rationale rebut methodological and theoretical criticisms leveled against our prior work. High pKa values for unconjugated bilirubin dictate that: a) bilirubin diacid, which readily diffuses across membranes and can cause neurotoxicity, is the dominant unbound bilirubin species of unconjugated bilirubin in plasma at physiological pH; b) at the near-neutral pH range of gallbladder bile, the monoanion is the major unconjugated bilirubin anion present, concordant with the finding that the calcium bilirubinate precipitated in gallstones is the monoanion salt. Our conclusions are thus relevant to understanding bilirubin-induced neurological disease in severely jaundiced neonates and the precipitation of calcium bilirubinate salts in gallstones.

Multidrug resistance associated protein 1 protects against bilirubin-induced cytotoxicity.

We have shown that multidrug resistance associated protein 1 (MRP1) mediates ATP-dependent extrusion of bilirubin, possibly limiting its potentially toxic accumulation in cells. To determine directly if Mrp1 protects cells against unconjugated bilirubin (UCB) toxicity, mouse embryo fibroblasts (MEF) were isolated from Mrp1 knockout (-/-) mice and their wild type (WT) (+/+) littermates. Compared to WT cells, cultured MEF (-/-) cells exposed to 40-140 nM unbound [H3]-bilirubin accumulated twice as much [H3]-bilirubin (P<0.01). This was associated with greater, dose-related cytotoxicity, assessed by the methylthiazoletetrazolium test, lactate dehydrogenase release and cellular ATP content. The data confirm that Mrp1 limits intracellular accumulation of UCB and thus decreases its cytotoxicity.

Bilirubin and the risk of common non-hepatic diseases.

Bilirubin is a potent antioxidant but can be toxic at high concentrations. This article critically reviews the reported relationships of plasma bilirubin levels to the severity and/or incidence of various common non-hepatic diseases. Plasma bilirubin levels are reportedly negatively related to the risk of atherosclerotic diseases, cancers, demyelinating neuropathies and seasonal affective disorder. By contrast, the incidence and severity of schizophrenia are increased by elevated bilirubin levels. The data strongly suggest that the level of plasma bilirubin should be considered as a risk factor for several common non-hepatic diseases. Additional studies are needed to clarify the mechanisms of this influence, which are thought to be related to unconjugated bilirubin counteracting the oxidative stress underlying these disorders.

Mortality outcomes after busulfan-containing conditioning treatment and haemopoietic cell transplantation in patients with Gilbert's syndrome: a retrospective cohort study.

BACKGROUND: Gilbert's syndrome is a common inherited disorder of bilirubin metabolism, characterised by mild, unconjugated hyperbilirubinaemia. However, the effect of Gilbert's syndrome on the disposition of some drugs can lead to unexpected toxicity. We tested the hypothesis that patients undergoing myeloablative conditioning and haemopoietic cell transplantation would have different mortality outcomes depending on whether or not they had laboratory evidence of Gilbert's syndrome. METHODS: In this retrospective cohort study, we used clinical and laboratory data of patients who had haemopoietic cell transplantation from Jan 1, 1991, to Dec 31, 2011. Patients were included if they had received high-dose conditioning regimens of cyclophosphamide plus total body irradiation (CY/TBI), busulfan plus cyclophosphamide (BU/CY), busulfan plus melphalan plus thioTEPA (BUMELTT), or melphalan before transplant. Patients were excluded if their original consent forms to report transplant outcomes were not signed, if consent was withdrawn, or if they were a prisoner. Patients with Gilbert's syndrome were defined as having laboratory values before the start of conditioning therapy for unconjugated serum bilirubin concentrations of at least 17·1 µmol/L (≥1 mg/dL), normal conjugated serum bilirubin, and no evidence of hepatitis, cholestasis, or haemolysis. We assessed the association of Gilbert's syndrome with overall mortality and non-relapse mortality using adjusted Cox regression models at day 200 after transplantation. FINDINGS: Our study cohort was 3379 patients-1855 (55%) allograft and 1524 (45%) autograft recipients. 211 (6%) patients had Gilbert's syndrome and 3168 (94%) did not have this condition. Most patients were adults (median age 45·8 years [IQR 33·2-55·5]) with haematological malignancies. For overall mortality 664 (20%) patients had died by day 200 after transplant (47 [22%] of 211 who had Gilbert's syndrome vs 617 [19%] of 3168 who did not have Gilbert's syndrome), and for non-relapse mortality 499 (92%) patients had died before relapse was recorded (38 [18%] who had Gilbert's syndrome vs 461 [15%] who did not have Gilbert's syndrome). The effect of Gilbert's syndrome on the risk of overall mortality and non-relapse mortality by transplant day 200 varied between the conditioning regimens and donor groups. In patients conditioned with a myeloablative regimen that contained busulfan (n=1131), those with Gilbert's syndrome (n=60) were at a significantly increased risk of death and non-relapse mortality by day 200 compared with those without Gilbert's syndrome (n=1071; hazard ratio [HR] 2·30, 95% CI 1·47-3·61, p=0·00030; and 2·77, 1·71-4·49, p<0·0001). In patients who received CY/TBI or melphalan conditioning regimens, those with Gilbert's syndrome had similar outcomes to those without Gilbert's syndrome (overall mortality at day 200 HR 0·90, 95% CI 0·60-1·34, p=0·60; non-relapse mortality at day 200: 0·90, 0·56-1·45, p=0·65). Analyses of causes of death and busulfan disposition provided no mechanistic explanation for the differences in mortality. INTERPRETATION: Overall mortality and non-relapse mortality at day 200 after transplant were significantly worse in patients with Gilbert's syndrome who received busulfan-containing myeloablative conditioning regimens, compared with non-Gilbert's syndrome patients. Patients with Gilbert's syndrome should receive busulfan-containing myeloablative conditioning regimens with caution. FUNDING: US National Institutes of Health.

Molecular basis of bilirubin-induced neurotoxicity.

Unconjugated bilirubin (UCB), at slightly elevated unbound concentrations, is toxic to astrocytes and neurons, damaging mitochondria (causing impaired energy metabolism and apoptosis) and plasma membranes (causing oxidative damage and disrupting transport of neurotransmitters). Accumulation of UCB in the CSF and CNS is limited by its active export, probably mediated by MRP1/Mrp1 present in choroid plexus epithelia, capillary endothelia, astrocytes and neurons. Upregulation of MRP1/Mrp1 protein levels by UCB might represent an important adaptive mechanism that protects the CNS from UCB toxicity. These concepts could explain the varied susceptibility of newborns to bilirubin neurotoxicity and the occurrence of neurological damage at plasma UCB concentrations well below therapeutic guidelines, and are relevant to the increasing prevalence of bilirubin encephalopathy in newborns.

The human multidrug-resistance-associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin.

Results of previous studies have suggested that UCB (unconjugated bilirubin) may be transported by MRP1/Mrp1 (multidrug-resistance-associated protein 1). To test this hypothesis directly, [3H]UCB transport was assessed in plasma-membrane vesicles from MDCKII cells (Madin-Darby canine kidney II cells) stably transfected with human MRP1 or MRP2; wild-type MDCKII cells served as controls. As revealed by Western blotting, transfection achieved abundant expression of MRP1 and MRP2. [3H]UCB uptake was measured in the presence of 60 microM human serum albumin at a free (unbound) concentration of UCB (B(F)) ranging from 5 to 72 nM and in the presence of 3 mM ATP or 3 mM AMP-PCP (adenosine 5'-[beta,gamma-methylene]triphosphate). MRP1-transfected vesicles showed transport activity three and five times higher respectively compared with MRP2 or wild-type vesicles, whose transport did not differ significantly. [3H]UCB transport was stimulated 4-fold by 1.5 mM GSH, occurred into an osmotically sensitive space, was inhibited by 3 microM MK571 and followed saturative kinetics with K(m)=10+/-3 nM (B(F)) and V(max)=100+/-13 pmol x min(-1) x (mg of protein)(-1). UCB significantly inhibited the transport of LTC4 (leukotriene C4), a leukotriene substrate known to have high affinity for MRP1. Collectively, these results prove directly that MRP1 mediates ATP-dependent cellular export of UCB and supports its role in protecting cells from bilirubin toxicity.

Low solubility of unconjugated bilirubin in dimethylsulfoxide--water systems: implications for pKa determinations.

BACKGROUND: Aqueous pKa values of unconjugated bilirubin are important determinants of its solubility and transport. Published pKa data on an analog, mesobilirubin-XIIIalpha, studied by 13C-NMR in buffered solutions containing 27 and 64 vol% (C2H3)2SO because of low aqueous solubility of mesobilirubin, were extrapolated to obtain pKa values in water of 4.2 and 4.9. Previous chloroform-water partition data on bilirubin diacid led to higher estimates of its pKa, 8.12 and 8.44, and its aqueous solubility. A thermodynamic analysis, using this solubility and a published solubility in DMSO, suggested that the systems used to measure 13C-NMR shifts were highly supersaturated. This expectation was assessed by measuring the residual concentrations of bilirubin in the supernatants of comparable DMSO-buffer systems, after mild centrifugation to remove microprecipitates. RESULTS: Extensive sedimentation was observed from numerous systems, many of which appeared optically clear. The very low supernatant concentrations at the lowest pH values (4.1-5.9) were compatible with the above thermodynamic analysis. Extensive sedimentation and low supernatant concentrations occurred also at pH as high as 7.2. CONCLUSIONS: The present study strongly supports the validity of the aqueous solubility of bilirubin diacid derived from partition data, and, therefore, the corresponding high pKa values. Many of the mesobilirubin systems in the 13C-NMR studies were probably supersaturated, contained microsuspensions, and were not true solutions. This, and previously documented errors in pH determinations that caused serious errors in pKa values of the many soluble reference acids and mesobilirubin, raise doubts regarding the low pKa estimates for mesobilirubin from the 13C-NMR studies.

Modulation of Mrp1 (ABCc1) and Pgp (ABCb1) by bilirubin at the blood-CSF and blood-brain barriers in the Gunn rat.

Accumulation of unconjugated bilirubin (UCB) in the brain causes bilirubin encephalopathy. Pgp (ABCb1) and Mrp1 (ABCc1), highly expressed in the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) respectively, may modulate the accumulation of UCB in brain. We examined the effect of prolonged exposure to elevated concentrations of UCB on expression of the two transporters in homozygous, jaundiced (jj) Gunn rats compared to heterozygous, not jaundiced (Jj) littermates at different developmental stages (2, 9, 17 and 60 days after birth). BBB Pgp protein expression was low in both jj and Jj pups at 9 days (about 16-27% of adult values), despite the up-regulation in jj animals (2 and 1.3 fold higher than age matched Jj animals at P9 and P17-P60, respectively); Mrp1 protein expression was barely detectable. Conversely, at the BCSFB Mrp1 protein expression was rather high (60-70% of the adult values) in both jj and Jj at P2, but was markedly (50%) down-regulated in jj pups starting at P9, particularly in the 4(th) ventricle choroid plexuses: Pgp was almost undetectable. The Mrp1 protein down regulation was accompanied by a modest up-regulation of mRNA, suggesting a translational rather than a transcriptional inhibition. In vitro exposure of choroid plexus epithelial cells obtained from normal rats to UCB, also resulted in a down-regulation of Mrp1 protein. These data suggest that down-regulation of Mrp1 protein at the BSCFB, resulting from a direct effect of UCB on epithelial cells, may impact the Mrp1-mediated neuroprotective functions of the blood-cerebrospinal fluid barrier and actually potentiate UCB neurotoxicity.

Effect of bilirubin on cytochrome c oxidase activity of mitochondria from mouse brain and liver.

BACKGROUND: The unbound, free concentration (Bf) of unconjugated bilirubin (UCB), and not the total UCB level, has been shown to correlate with bilirubin cytotoxicity, but the key molecular mechanisms accounting for the toxic effects of UCB are largely unknown. FINDINGS: Mouse liver mitochondria increase unbound UCB oxidation, consequently increasing the apparent rate constant for unbound UCB oxidation by HRP (Kp), higher than in control and mouse brain mitochondria, emphasizing the importance of determining Kp in complete systems containing the organelles being studied. The in vitro effects of UCB on cytochrome c oxidase activity in mitochondria isolated from mouse brain and liver were studied at Bf ranging from 22 to 150 nM. The results show that UCB at Bf up to 60 nM did not alter mitochondrial cytochrome c oxidase activity, while the higher concentrations significantly inhibited the enzyme activity by 20% in both liver and brain mitochondria. CONCLUSIONS: We conclude that it is essential to include the organelles being studied in the medium used in measuring both Kp and Bf. A moderately elevated, pathophysiologically-relevant Bf impaired the cytochrome c oxidase activity modestly in mitochondria from mouse brain and liver.

Bilirubin chemistry and metabolism; harmful and protective aspects.

Unconjugated bilirubin (UCB), the principal mammalian bile pigment, is the end product of heme catabolism. Both belong to the superfamily of tetrapyrrolic compounds that serve multiple biological functions in animals and plants. Its six internal hydrogen bonds give UCB a unique structure responsible for its physico-chemical properties and biological effects. Like many weakly-polar, poorly-soluble compounds, UCB is transported in blood tightly bound to albumin, with less than 0.01% of total bilirubin circulating in an unbound form (free bilirubin, Bf). This fraction governs the diffusion of UCB into tissues, and therefore Bf is responsible for both its beneficial and toxic effects on cells. Although, UCB was long thought to be a non-functional waste product, recent studies have shown that the antioxidant effects of mildly elevated serum bilirubin levels, as well as activation of heme oxygenase, may protect against diseases associated with oxidative stress, such as atherosclerosis. By contrast, markedly elevated serum UCB levels may cause severe neurological damage, especially in neonates. The regulation of cellular UCB content, by its conjugation, oxidation, and export, are, therefore of paramount importance to cellular health.