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Bilirubin is a potent antioxidant generated intracellularly during the degradation of heme by the enzyme heme oxygenase. The purpose of this study was to determine the role of increased cardiac bilirubin in protection against postischemic myocardial dysfunction. Rat hearts were isolated and perfused according to the Langendorff technique to evaluate the recovery of myocardial function after 30 min of global ischemia and 60 min of reperfusion. We found that upregulation of the inducible isoform of heme oxygenase (HO-1) by treatment of animals with hemin 24 h before ischemia ameliorated myocardial function and reduced infarct size (tetrazolium staining) on reperfusion of isolated hearts. Tin protoporphyrin IX, an inhibitor of heme oxygenase activity, completely abolished the improved postischemic myocardial performance observed after hemin-mediated HO-1 induction. Likewise, cardiac tissue injury was exacerbated by treatment with tin protoporphyrin IX. Increased cardiac HO-1 expression and heme oxygenase activity were associated with enhanced tissue bilirubin content and an increased rate of bilirubin release into the perfusion buffer. Furthermore, exogenously administered bilirubin at concentrations as low as 100 nanomolar significantly restored myocardial function and minimized both infarct size and mitochondrial damage on reperfusion. Our data provide strong evidence for a primary role of HO-1-derived bilirubin in cardioprotection against reperfusion injury.

Bilirubin is taken up from blood into hepatocytes by sinosuidal membrane transporters and then excreted into bile through the bile canalicular membrane mainly as bilirubin glucuronides. (1) Mechanism of bilirubin uptake into hepatocytes: many organic anions are incorporated into hepatocytes by organic anion transporting polypeptides (rat, oatp1, oatp2, oatp3; human, OATP), liver-specific transporter (rlst/HLST), and/or by organic anion transporters (OAT2, OAT3). Oatp1 and HLST transport bilirubin monoglucuronide. However, a transporter of unconjugated bilirubin in the sinusoidal membrane has not as yet been identified. Unconjugated bilirubin may also go across the hepatocyte sinusoidal membrane by a diffusion process. (2) Intrahepatic transport and conjugation of bilirubin: ligandin carries bilirubin to the endoplasmic reticulum (ER) of hepatocytes. In the ER, bilirubin is conjugated by bilirubin uridine diphosphate (UDP)-glycosyltransferase (bilirubin UGT; UGT1A1) to form mono- and diglucuronides of bilirubin. (3) Transport mechanism of bilirubin glucuronides across the hepatocyte canalicular membrane: at the canalicular membrane, bilirubin glucuronides are excreted into bile by multidrug resistance-associated protein 2 (MRP2), a member of the ATP-binding cassette transporter family. (4) Regurgitation of bilirubin glucuronides into blood: MRP3, which is located in the lateral membrane, transports bilirubin glucuronides into blood under conditions of impaired biliary bilirubin excretion.

One of the most remarkable revolutions during the history of animal kingdom is the adaptation to oxygen, a highly toxic gas produced by plants. Based on the high affinity of heme toward oxygen, it has been believed that heme itself and/or hemoprotein should play significant roles in the processes of adaptation. Recently, two novel functions of heme and its metabolites were identified; namely, hemoprotein is an oxygen sensor and biliverdin and bilirubin, catabolites of heme, are antioxidants. Thus, heme is a key molecule in the responses to environmental stress, including oxygen. Although activation of hypoxia inducible factor-1, which induces expression of genes encoding erythropoietin and heme oxygenase (HO-1), is generally accepted to be controlled by oxygen sensor, the precise signaling pathway has not yet been well elucidated. Various stresses such as hypoxia are known to activate the HO-1 gene, the key enzyme of heme degradation, resulting in the marked conversion of pro-oxidant (heme) into its antioxidative catabolites. The induction is, therefore, supposed to have protective effects. Recent reports on HO-1 deficiency also support this hypothesis that the activation of the HO-1 gene is one of the most important defense mechanisms against environmental stress.

This study aimed to examine whether an elevated activity of heme oxygenase (HO)-1 in the tissue attenuates endothelial cell-leukocyte interactions microvessels in vivo. When rats were pretreated with an intraperitoneal injection of hemin, an HO-1 inducer, mesenteric tissues, including their microvessels, displayed a marked induction of HO-1 concurrent with an increase in plasma concentrations of bilirubin-IXalpha, the product of HO-catalyzed degradation of protoheme IX. In these rats, oxidative stress such as superfusion with H(2)O(2) and ischemia-reperfusion of the tissues neither induced rolling nor exhibited adherent responses of leukocytes in venules. In contrast, the oxidative stresses evoked marked rolling and adhesion of leukocytes in the control rats without HO-1 induction. The HO-1 induction also downregulated leukocyte adhesion elicited by other pro-oxidant stimuli such as N(omega)-nitro-L-arginine methyl ester. The decreases in the oxidant-elicited leukocyte adhesive responses under HO-1-inducing conditions were restored by perfusion with zinc protoporphyrin-IX, an HO inhibitor, but not with copper protoporphyrin-IX, which did not inhibit the enzyme. Furthermore, the effects of zinc protoporphyrin-IX were repressed by superfusion with bilirubin or biliverdin at the micromolar level, but not by the same concentration of carbon monoxide, another product of HO. These results indicate that induction of the HO-1 activity serves as a potential stratagem to prevent oxidant-induced microvascular leukocyte adhesion through the action of bilirubin, a product of HO reaction.

Heme oxygenase (HO) catalyzes the conversion of heme to carbon monoxide, iron, and biliverdin, which is immediately reduced to bilirubin (BR). Two HO active isozymes exist: HO1, an inducible heat shock protein, and HO2, which is constitutive and highly concentrated in neurons. We demonstrate a neuroprotective role for BR formed from HO2. Neurotoxicity elicited by hydrogen peroxide in hippocampal and cortical neuronal cultures is prevented by the phorbol ester, phorbol 12-myristate 13-acetate (PMA) via stimulation of protein kinase C. We observe phosphorylation of HO2 through the protein kinase C pathway with enhancement of HO2 catalytic activity and accumulation of BR in neuronal cultures. The neuroprotective effects of PMA are prevented by the HO inhibitor tin protoporphyrin IX and in cultures from mice with deletion of HO2 gene. Moreover, BR, an antioxidant, is neuroprotective at nanomolar concentrations.

Atherosclerosis is a major contributor to cardiovascular disease, and genetic disorders of lipoprotein metabolism are recognized risk factors in atherogenesis. The gaseous monoxides nitric oxide (NO) and carbon monoxide (CO), generated within the blood vessel wall, have been identified as important cellular messengers involved in the regulation of vascular smooth muscle tone. Microsomal heme oxygenases degrade heme to biliverdin and CO, and the cytosolic enzyme biliverdin reductase then catalyzes reduction of biliverdin to bilirubin, both powerful chain-breaking antioxidants. Two principal isozymes of heme oxygenase have been identified, a constitutive isoform HO-2 (M(r) approximately 34,000) and an inducible isoform HO-1 (M(r) approximately 32,000), which is expressed at a low basal level in vascular endothelial and smooth muscle cells and is induced by heavy metals, oxidative stress, inflammatory mediators and oxidized low density lipoproteins. Although NO and CO modulate intracellular cGMP levels, platelet aggregation and smooth muscle relaxation, CO has a much lower affinity for soluble guanylyl cyclase than NO. Decreased production or sensitivity to NO in atherosclerosis may be compensated for by an induction of HO-1, with bilirubin acting as a cellular antioxidant and CO as a vasodilator. This review examines the evidence that oxidized low density lipoproteins (LDL), hypoxia and pro-inflammatory cytokines induce HO-1 expression and activity in vascular endothelial and smooth muscle cells, and evaluates the anti-atherogenic potential of the heme oxygenase signalling pathway.

Background: We propose a modification of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method to study the cytotoxicity of bilirubin. The original method involves reading the intensity of a purplish blue color resulting from the conversion of MTT to formazan crystals by the mitochondria of viable cells. We have found that when the method is applied to study the effect of bilirubin on growing cells, precipitation of the yellow bilirubin pigment interferes with the colorimetric reading., Methods: A human liver cell line was used. The interference of bilirubin deposition on the MTT assay was investigated by comparing the value of optical density of the MTT solution in the presence and absence of bilirubin. The effect of 0.04 mol/L HCL-isopropanol on the bilirubin precipitate was tested by recovering the amount of bilirubin from the wells after the isopropanol treatment., Results: Bilirubin deposition increases MTT reading by 10-24%. Hydrochloride-isopropanol (0.04 mol/L) dissolves MTT formazan only without disturbing the bilirubin precipitates. The bilirubin extracted into the supernatant was less than 5% of the total bilirubin deposited., Discussion: This indirect MTT assay, as developed in this study, could eliminate the interference of bilirubin deposits and serve as a good method for the study of bilirubin cytotoxicity.

Rapid growth of recurrent disseminated tumor and/or metastases can occur in children after incompletely resected, non-pretreated malignant hepatoblastoma (HB). This accelerated tumor growth is observed during the first four postoperative weeks, which is the period of maximal liver regeneration. One key regulator of regeneration, the hepatocyte growth factor (HGF), may be responsible for the induction of tumor growth. We, therefore, investigated levels and sources of HGF in HB patients. With ELISA we measured elevated serum levels (> 1,000 pg/ml) of HGF in 10 of 23 HB patients in comparison with three healthy children (< 610 pg/ml). HGF values of non-pretreated children with HB ranged from 169-10,183 pg/ml (mean 889 pg/ml) while those of patients after primary chemotherapy reached 608-15,000 pg/ml (mean 4,556 pg/ml). An up to fourfold increase of HGF was detected in 10 of 12 children 24-72 hours after liver resection. With immunoenzymatic staining on cryostat sections and cytospin preparations of the tumors we could localize HGF to the fibroblasts of the mesenchymal tumor components. In contrast, its receptor (c-met) was found to be expressed on the epithelial HB cells. Our results indicate that HGF secretion is enhanced after liver resection in children with HB and thus could have a biological function for growth of HGF receptor-expressing tumor cells. The results of immunostaining further suggest that HB is able to produce HGF as its own growth factor in a local paracrine fashion.

Background: Large intravenous bilirubin loads cause loss of hepatic canalicular membrane microvilli and cholestasis. This study examines whether these untoward effects might be due to canalicular membrane injury from cytotoxic bile., Methods: The cytotoxicity of bile was assayed against pig erythrocytes before and throughout 4.5-h intravenous infusion of 170 microg kg(-1) body weight of bilirubin in anaesthetized pigs. The capacity to generate canalicular bile flow was tested before and after bilirubin infusion by means of short-term intraportal cholic acid infusion., Results: Bilirubin infusion increased the cytotoxicity of hepatic bile, reduced biliary phospholipid secretion by 90%, and caused cholestasis. Cholic acid infusion before bilirubin also increased the cytotoxicity of bile but increased bile flow and doubled biliary phospholipid output., Conclusion: Large intravenous bilirubin infusions increase the cytotoxicity of bile, suppress biliary phospholipid secretion, and render hepatic canalicular membrane microvilli susceptible to injury from cytotoxic bile so that cholestasis occurs.

Protein phosphorylation is an important mechanism for regulation of cell processes. Bilirubin inhibits phosphorylation of several peptides/proteins by a number of different kinases, and this may contribute to the toxic effects of bilirubin on cells, particularly neurons. Bilirubin binds to lysine residues on both albumin and ligandin. The ATP-binding subdomain II on protein kinases contains an invariant lysine, which might hypothetically be involved in mediation or modulation of bilirubin-inhibition of protein phosphorylation. We have studied the ability of lysine-containing peptides to modulate the effects of bilirubin, using phosphorylation of a phospholemman peptide catalyzed by protein kinase A as a model system. Addition of bilirubin (50 microM) decreased the activity of the catalytic subunit of protein kinase A by 75%. A synthetic lysine-containing decapeptide which mimicked part of subdomain II on the protein kinase family was partially able to prevent the bilirubin effects. Similar effects were not observed with two other decapeptides in which lysine had been replaced by arginine or alanine. Polylysine (100 microM) completely prevented the inhibitory effect of 50 microM bilirubin, whereas polyglutamate and polyarginine did not have this effect. Poly-D-lysine and poly-L-lysine appeared to be equivalent in their ability to prevent the bilirubin effect. These data support the notion that binding of bilirubin to lysine may play a role in the mediation and/or modulation of bilirubin neurotoxicity.

Heme oxygenase the rate-limiting step in the degradation of heme to bilirubin, generates carbon monoxide. This gaseous molecule plays important roles in neuronal signaling and modulation of vascular tone. Additionally, carbon monoxide is involved in some pathological conditions (e.g., ischemia, endotoxic shock, excitotoxicity) as a protective or toxic factor. Bilirubin, another heme metabolite, exhibits intriguing biological activities as an antioxidant, an antimutagen, and an anti-complement agent. Vital functions and the dual nature displayed by these two heme metabolites are discussed.

The prevailing hypothesis for phototransduction is that visual (rod or cone) pigments mediate light's primary effects on biological clock systems. Common light-responsive chronobiological behavioral properties of plants and animals and some common molecular structures of plants and animals suggest the possibility that heme moieties and bile pigments in animals mediate some nonvisual influences of light on neuroactive gases and biological rhythms. As plant phytochrome resets the plant biological clock, the similar chromophore in bile pigments is proposed to transduce environmental light zeitgeber signals to endogenous biological clocks. The temporal association of plasma bilirubin and rapid-eye-movement (REM) sleep in populations, the correlation of secretion of biliary bilirubin with REM sleep among 10 different species (Spearman r = 0.89, p < 0.002), and the known responses of bilirubin to light lead to the hypothesis that bilirubin, in particular, plays an evolutionary role in the regulation of REM sleep and in mediating some of light's antidepressant effects.

Objective: To review the literature on transcutaneous bilirubinometry so that its exact role in the prevention of kernicterus or bilirubin encephalopathy could be determined., Design and Methods: Literature searches were done in Medline and Current Contents., Results: It is estimated that about 50% of newborns have an episode of jaundice in the first few days of life. Six percent of newborns may develop hyperbilirubinemia (> 220 mumol/L), which can potentially cause bilirubin encephalopathy or kernicterus, a severe neonatal disease. In the past, serum bilirubin (SB) has been the preferred method of detecting hyperbilirubinemia in newborns. The ordering of SB in neonates is based on visual evaluation by either physicians or nursing staff. Skin puncture collection of blood exposes the neonate to trauma and risk of infection. A noninvasive device for predicting serum bilirubin levels in newborns diminishes the need to do skin punctures. One such device that has been very extensively studied is the Minolta AirShields Jaundice Meter. It is a portable light-weight instrument that uses reflectance measurements on the skin to determine the amount of yellow color present in the skin, namely transcutaneous bilirubin (TcB). Although the TcB measurements correlate well with serum bilirubin (SB) levels, they cannot accurately predict serum bilirubin because of error related to a variety of factors., Conclusions: TcB cannot be used directly to make decisions about transfusions or phototherapy in neonates. It is a good tool for screening neonates to determine when a laboratory measurement of serum bilirubin is needed. Such a practice requires careful selection of the decision level so that false-negative TcB values do not prevent appropriate serum bilirubin tests from being done.

In vitro, bilirubin is a strong antioxidant, but in vivo its capacity to act as a scavenger of toxic oxygen radicals remains poorly documented. The aim of this study was to evaluate of bilirubin had antioxidant properties in jaundiced infants. The antioxidant capacity of neonatal plasma was measured in Trolox equivalents (TEAC, mmol/l) and correlated in vitro with plasma bilirubin concentrations (r2 = 0.99). Plasma TEAC was compared before and after exchange transfusions for neonatal hyperbilirubinemia (250-435 mumol/l). The antioxidant properties of the paired blood samples before and after exchange transfusions (TEAC: 1.67 +/- 0.12 vs. 1.37 +/- 0.09 mmol/l, n = 11) varied in proportion to the serum bilirubin levels. The changes in other antioxidants were not large enough to account for the magnitude of change in antioxidant capacity. Therefore, in vivo, the plasma antioxidant capacity of jaundiced newborn infants is related to the level of bilirubin.

The anti-oxidative effect of bilirubin was investigated in an ischemia-reperfusion model of rat liver. The rat portal vessel and liver artery were ligated with a vascular clip, and after reperfusion the urine was collected at intervals. The amount of biopyrrins, the oxidative metabolites of bilirubin, in rat urine reached a maximum 4 hours after reperfusion. Biotripyrrin-a and -b which are biopyrrins isolated from human urine were included in urinary bilirubin oxidative metabolites of rats after reperfusion. The hepatic mRNA level of heme oxygenase-1 (HO-1), the rate limiting enzyme of bilirubin biosynthesis, reached a maximum after 4 hours. Furthermore, the hepatic activity of HO began to rise 4 hours after treatment and remained high until 24 hours posttreatment. These findings suggest that bilirubin acts as a physiological antioxidant in vivo in ischemia-reperfusion and that bilirubin biosynthesis is evoked by oxidative stress.

Background & Aims: Patients with ileal disease, resection, or bypass are at increased risk of developing pigment gallstones, but the pathophysiological mechanisms are unknown. The aim of this study was to test the hypothesis that ileectomy induces enterohepatic cycling of bilirubin., Methods: Ileectomy or sham operation was performed in adult male Sprague-Dawley rats with the following control procedures: no operation, ileal transection, proximal or distal jejunectomy, ileocolonic transposition, and ileocecectomy. Bilirubin and bile salt secretion rates were measured after bile duct cannulation performed 3-11 days after intestinal surgery. Also measured were bilirubin and bile salt concentrations in the colon as well as indices of hemolysis in blood., Results: Compared with controls, bilirubin secretion rates were increased significantly 3-5 days after ileectomy, distal jejunectomy, ileocolonic transposition, and ileocecectomy, with no hemolysis occurring. Bile salt secretion rates also increased significantly after ileectomy but decreased markedly with prevention of coprophagy, whereas bilirubin secretion rates remained elevated. By 8-11 days after surgery, intestinal adaptation normalized bile salt reabsorption, and hypersecretion of bilirubin was abolished. Colonic levels of unconjugated bilirubin and bile salts were increased fivefold and eightfold respectively in ileectomized animals, but unconjugated bilirubin levels remained normal in bile., Conclusions: These results are consistent with the hypothesis that enterohepatic cycling of bilirubin occurs with bile salt malabsorption.

We examined the possibility that bilirubin physiologically acts as an antioxidant by using scurvy-prone ODS-od/od rats treated with endotoxin (lipopolysaccharide: LPS). Recently, bilirubin oxidative metabolites were isolated from human urine and named biotripyrrin-a and biotripyrrin-b. The LPS injection markedly increased bilirubin oxidative metabolites in urine of rats fed an ascorbic acid-free diet. This increase was supressed by feeding an adequate amount of ascorbic acid, a physiological antioxidant. the concentrations of biotripyrrin-a and -b in urine collected 6.5-10 h after the LPS injection were lower in rats fed an ascorbic acid-supplemented diet than in rats fed an ascorbic acid-free diet. Moreover, feeding with ascorbic acid suppressed the elevation of hepatic mRNA level of heme oxygenase-1, the rate-limiting enzyme of bilirubin biosynthesis, in rats injected with LPS. These findings suggest that bilirubin is oxidized in rats treated with LPS and acts as a physiological antioxidant synergistically with ascorbic acid in vivo.

The manifestations of bilirubin encephalopathy include disturbances in the visual pathway (visual gaze paralysis and distorted visual perception). In the young jaundiced Gunn rat (jj) model of hyperbilirubinemia, significant differences in visual evoked potential (VEP) patterns have been recorded during development. In the present study, the effects of sulfadimethoxine (SDM) on VEP and electroretinogram (ERG) were examined in 3-wk-old jj rats. This drug displaces bilirubin from its albumin binding sites in the circulation, shifting it into tissues including the brain. Marked latency prolongations (11-20%) and reduced amplitudes (20-64%) were observed in the different wave components of the VEP. These changes were evident as early as 2 h after injection of the drug and persisted thereafter for another 4 h. On the other hand, ERG changes (significant prolongation of wave b) became apparent in these animals only 6 h after SDM injection. These results suggest that, although some changes in the retina may occur after a massive entry of bilirubin into the nervous system, the primary damage in the visual pathway after bilirubin exposure is probably beyond the retina.

Cerebral arteries in spasm have been found to contain low levels of adenosine triphosphate (ATP), and it has been postulated that this change in levels results from hypoxia produced by arterial encasement in clotted material. This study was undertaken to determine whether any of four blood-derived agents, ferrous hemoglobin, methemoglobin, hemin, or bilirubin, is capable of reducing energy levels in cerebral artery smooth-muscle cells. Twenty-four-hour exposure of cultured canine basilar artery cells to ferrous hemoglobin and bilirubin led to a significant decline in ATP levels (to 8.9 nmol/mg protein and 2.8 nmol/mg protein, respectively) versus control (16.6 nmol/mg protein); methemoglobin and hemin showed no effect. Bilirubin but not hemoglobin was found to interfere with electron transport and with creatine phosphokinase activity in intact cells; however, bilirubin showed no inhibitory effect on this enzyme in cell-free conditions. The findings indicate that hemoglobin and bilirubin may be responsible for diminished energy levels in cerebral arteries. These observations also suggest that bilirubin may exert its effect on ATP by impairing mitochondrial function.

In this study, using flow cytometry, we investigated the autofluorescence emitted by lymphocytes, monocytes and neutrophils exposed to different unconjugated bilirubin concentrations and investigated the relationship between these parameters. Different unconjugated bilirubin concentrations were prepared from a newborn serum with an unconjugated bilirubin concentration of 800 m mumol/l. The same concentrations of unconjugated bilirubin have been prepared from pure bilirubin. 10 microliters of cord blood were incubated at room temperature for 15 min with 90-microliter solutions with different bilirubin concentrations prepared from both serum and pure bilirubin. After incubation, cells were washed three times with PBS, erythrocytes were lysed by lysing buffer and run through flow cytometry immediately. Autofluorescence was measured by recording mean fluorescence channels for lymphocytes, monocytes and neutrophils. There was a good correlation between serum concentrations of unconjugated bilirubin that cells were exposed to and the autofluorescence intensity of neutrophils (r = 0.904, p < 0.005), monocytes (r = 0.759, p < 0.05) and lymphocytes (r = 0.766, p < 0.01). Results obtained with pure bilirubin were also similar. Autofluorescence emitted by lymphocytes was lower than that of monocytes (p < 0.01) or neutrophils (p < 0.0005).

Cobalt chloride (CoCl2), a well-known inducer of heme oxygenase, produced a strong increase of in vivo rat liver chemiluminescence (QLV) 6 h after its administration. The activity of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) was found to be significantly decreased 9 h after CoCl2 injection. Heme oxygenase activity increased 9 h after treatment, reaching a maximum value around 18 to 24 h after CoCl2 administration. This induction was preceded by a decrease in the intrahepatic GSH pool and an increase in hydrogen peroxide steady state concentration, both effects taking place several hours before induction of the heme-oxygenase. Co-administration of Sn-protoporphyrin IX, a potent inhibitor of heme oxygenase, completely prevented the enzyme induction, increasing the QLV levels. Administration of bilirubin, the end product of heme catabolism in mammals, prevented the heme oxygenase induction as well as the decrease in hepatic GSH and the increase of chemiluminescence when it was administered 2 h before CoCl2 treatment. These results support the proposal that the induction of heme oxygenase by cobalt chloride may be a general response to oxidant stress and, by increasing bilirubin levels, could constitute an important cellular defense mechanism against oxidative damage.

We hypothesized that because bilirubin is a potent free-radical quencher, infants without disorders that have oxygen-radical disease (ORD)-mediated mechanisms may have higher bilirubin levels than infants suffering from conditions possibly associated with ORD-mediated mechanisms (e.g., necrotizing enterocolitis, broncopulmonary dysplasia, intraventricular hemorrhage, and retinopathy of prematurity). We identified 25 infants (birth weight 912 +/- 208 gm, gestational age 27 +/- 3 weeks) who comprised the ORD group and compared them with 57 controls (birth weight 1242 +/- 248 gm, gestational age 31 +/- 3 weeks). Infants with ORD had lower peak serum bilirubin concentrations, later ages at peak, and lower incidence of peak bilirubin concentrations exceeding 10 or 15 mg/dl. In addition, these infants exhibited a slower rate of bilirubin rise and a smaller area under the bilirubin-time curve measure compared with controls. To control for different birth weights, we analyzed subgroups weighing < 1000 gm. Significant differences were again identified in peak bilirubin concentrations, age at peak, phototherapy duration, and area under the curve. In this population of preterm infants, higher bilirubin levels were associated with a lower incidence of oxygen radical-mediated injury.

The fact that almost all neonates exhibit a "physiological" jaundice, prompts the question whether bilirubin, usually exclusively considered a potentially toxic endproduct of the metabolism of heme, might not also have a positive task in the first days of life. A recently discovered property of bilirubin under in vitro conditions is its ability to combine with free oxygen radicals such as are produced in the oxidative metabolic processes of the neonate immediately following birth. In the present article, the concept of the anti-oxidative effect of bilirubin, and its translation to the early neonatal period is presented and discussed on the basis of a number of examples.

The prevention of retinopathy of prematurity (ROP) remains a persistent problem. A previous report has focused on the possible protective effect of bilirubin on the development of ROP. These results still await clinical confirmation by other research groups. Therefore, we undertook a retrospective clinical study trying to confirm this attractive hypothesis. Twelve premature newborns under 32 weeks of gestation with ROP stage 3-4 were matched for gestational age with 12 infants without ROP. Data were collected about the infant's characteristics, medical illnesses, ventilatory settings and treatments. The total serum bilirubin concentrations between the 1st and 8th postnatal day were also gathered. The two matched groups were comparable as to their basic data, clinical characteristics and treatment, except for a slight, but significant longer duration of phototherapy for group ROP 0 (mean, 50.2 h; SD 48,6 vs 31.6 h; SD 42.7 in ROP 3-4; P = 0.02). No statistical difference relative to bilirubin was found between the two groups, neither when expressed as daily mean concentrations, nor as area under the curve (AUC) (mean, ROP 0: 17876.7; SD 6077.3 vs 18888.4; SD 55552.7 in ROP 3-4; P = 0.404) or AUC/h (mean, ROP 0: 135.1; SD 36.3 vs 144.1; SD 23.2 in ROP 3-4; P = 0.515). Our findings do not confirm the hypothesis of a clinically measurable, beneficial role of bilirubin on the development of ROP.

Oxidative damage to biological macromolecules has been implicated in a number of diseases. Much interest has focused on how non-proteinaceous, low-molecular weight antioxidants prevent oxidative damage to lipids, while comparatively little is known about protein antioxidation. Here we show that bilirubin (BR), the end-product of heme catabolism, when bound to bovine serum albumin (BSA), is oxidised by hydroxyl (.OH), hydroperoxyl (HO2.), and superoxide anion (O2-.) radicals to so far mostly uncharacterised products. The initial oxidation rates of BSA-bound BR decreased in the order OH > HO2. > O2-.. BR protected its carrier protein from oxidative damage inflicted by .OH radicals. This protective action included a reduction in the .OH-mediated cleavage of BSA, conversion of Trp into kynurenine and formation of 'bityrosine-specific' fluorescence. BR also strongly inhibited .OH-mediated formation of protein carbonyls, whereas ascorbate and Trolox (a water-soluble analogue of vitamin E) were much less effective. These results support an antioxidant-protective function of BR and point towards significant differences in the efficacies of various antioxidants in the prevention of oxidative damage to lipids and proteins.

In order to explore the substantial role of monoconjugated bilirubin (MCB) in gallstone formation, bile pigment precipitation and hemolytic jaundice, three experimental protocols have been studied, namely, (1) MCB and dietary induced pigment gallstone model, (2) MCB in human gallstone and incubated bile precipitates and (3) MCB in hemolytic jaundice. It was found that doubly increased MCB accounted for 1/3 of the total pigment in lithogenic guinea pig and CDCA plus glycine possessed certain protective effect from gallstone development; MCB was found in human gallstones both in bilirubinate and cholesterol type, and an unknown pigment, possibly an isomer of MCB, was found in black stone. During experimental hemolytic jaundice model preparation, both MCB and UCB were elevated, and MCB was found increased by 10 times, even exceeding the concentration of DCB when the injected bilirubin was about 4 mg/kg of body weight. It is reasonable to consider that MCB as a coprecipitant with UCB and a precursor of UCB played an essential role in the pathogenesis of gallstones.

More and more diseases have been proposed to have a radical or oxidant involvement. Although in most cases we do not know if this involvement is a cause or a result of the disease process, it is still valuable to learn about those compounds or enzymes that might block, inhibit, or prevent radical-initiated reactions. Therefore, it becomes increasingly important to understand which compounds can function as antioxidants, where they are located in the body, and what their mechanism of action might be. As we increase our knowledge in these areas, we will have a better opportunity to propose interventions that might suppress or even reverse some of the ravages of oxidant-based diseases in humans.

In order to investigate the influence of indirect bilirubin to O2-dependent bactericidal mechanism in adult and newborn phagocytes we use the NBT reduction activity of granulocytes and monocytes as index, by infiltrated granulocytes and monocytes in different concentration of indirect bilirubin with or without latex stimulator. The NBT reduction activity of granulocytes infiltrated in 20 mg/dl indirect bilirubin will increase slightly, compared to uninfiltrated; but with latex stimulator, the increment will decrease significantly, this change is reversible, when indirect bilirubin be washed, the stimulating capacity by latex stimulator in granulocyte will be stronger; there were no such change pattern in monocytes. Since there were a big difference in NBT reduction activity of granulocytes and monocytes between newborns, with or without latex stimulator, we can't get a satisfied result. Indirect bilirubin in cells will increase NBT reduction activity of monocytes, but not in granulocytes.

The effects of bile acids and bilirubin, which increase in blood in obstructive jaundice, on bicarbonate secretion were studied experimentally using isolated antral mucosa of the guinea pig. Antral mucosal preparations were mounted between Ussing chambers. Basal and bethanechol-stimulated secretion were measured by a pH stat device using 5 mM HCl. After 10(-4), 10(-3), and 5 x 10(-3) M taurocholic acid, 10(-4) and 10(-3) M cholic acid, and 1.2 x 10(-4) and 2.4 x 10(-4) M bilirubin conjugate were added to the serosal solution, basal and bethanechol-stimulated secretion were also measured. Taurocholic acid, at any dose, did not affect basal secretion, but bethanechol-stimulated secretion was inhibited dose dependently. Cholic acid and bilirubin conjugate did not affect basal secretion, but bethanechol-stimulated secretion was significantly inhibited. Although the addition of bile acid or bilirubin into the serosal solution under experimental conditions is not the same as obstructive jaundice, these studies suggest that the inhibition of bicarbonate secretion in the gastric mucosa may have an important role in the formation of acute gastric mucosal lesions in obstructive jaundice.

Endothelial cells cultured for 3 days in the presence of hemoglobin H pooled sera had significantly decreased cell proliferation compared to those in normal serum. Inhibition was demonstrated at a concentration of 20% pooled serum in the cultured medium. Further decrease was shown in the presence of 30% pooled hemoglobin H sera. Sera from two genotypes of Hb H disease (alpha-thal 1/alpha-thal 2 and alpha-thal 1/Hb Constant Spring) had the same degree of inhibitory effect. Pooled sera from beta-thal/Hb E patients (both splenectomized or nonsplenectomized cases) had no such inhibitory effect. However, at day 4 and 5, the growth pattern relatively declined. Bilirubin at a concentration greater than 4.0 mg% in the medium 199 also caused significant decrease in cell proliferation. Since the diluted Hb H serum had bilirubin less than 4.0 mg%, the inhibitory effect of the pooled HbH serum is thus not due to effect of bilirubin. The difference between HbH and beta-thal/HbE sera in terms of inhibition of endothelial cell proliferation is the new finding that needs further investigation to explain vascularization and hemostasis in the patients of these two genotypes.

The mothers of experimental neonates were administered excess bilirubin for a month, and the neonates were suffering from hyperbilirubinemia. The studies were conducted on the effect of excess bilirubin and metalloporphyrins on plasma membrane and mitochondrial membrane. We have isolated, separated, and estimated phospholipids, and also assayed the activity of phospholipase A2 from whole liver and mitochondrial and microsomal fractions. Excess of bilirubin administration decreased the total phospholipid level and inhibited the phospholipase A2 activity. Cr-PP (chromium protoporphyrin) induces the phospholipase A2 activity which is inhibited by simultaneous bilirubin administration. However, Zn-PP (zinc protoporphyrin) and Mn-PP (manganese protoporphyrin) showed a reverse pattern.

In neonatal hyperbilirubinaemia the distribution of unconjugated bilirubin (BR) between albumin and cellular binding determines its toxicity. The relationship between the ratio of BR/albumin and the impairment of cellular functions was studied employing a system of antibody-dependent cellular cytotoxicity (ADCC) with erythrocytes as target cells. The cytotoxic activity of peripheral blood monocytes, lymphocytes and granulocytes is inhibited in a concentration-related manner depending on the molar ratio BR/albumin. The occupation of albumin binding sites by BR is related to the extent of inhibition of ADCC activity suggesting that there is a competition between albumin and cellular binding sites for free bilirubin. This competition is governed by the respective affinity and concentration of the binding sites. Bilirubin binding to the cellular (silent) membrane receptors leads to the chain of events resulting in impairment of cellular functions, i.e. ADCC.

Previous studies have shown that intraperitoneal bile increases bacterial growth and mortality in Escherichia coli peritonitis in the rat. The purpose of the present study was to determine a) the influence of bile acids (cholic, deoxycholic, or chenodeoxycholic) and bilirubin on survival, bacterial growth, and superoxide release by peritoneal phagocytes in this model, and b) the effect of bile acids on bacterial growth and endotoxin release when incubated with E. coli in vitro. Each of the bile acids aggravated the E. coli peritonitis, with increased bacterial counts in the peritoneal cavity and in blood and increased mortality. Deoxycholic acid was the most deleterious of the bile acids, causing suppression of superoxide release by peritoneal phagocytes, like whole bile. In vitro, bile acids did not seem to affect growth of E. coli, but cholic and deoxycholic acid seemed to enhance the release of endotoxin. It is concluded that the bile acids are responsible for the noxious effect of bile in E. coli peritonitis. It is suggested that the detergent properties of bile acids aggravate the peritonitis by solubilizing the cell membranes of both bacteria and phagocytes.

Elevated levels of bilirubin and other tetrapyrroles are common to a number of chronic hematologic and liver diseases that can result in growth failure. This report establishes a cellular model system for the study of these endogenous growth inhibitors. Primary chondrocyte cultures were prepared from embryonic chick cartilage; cells were incubated (0.3 X 10(5) cells per plate) in tissue culture medium containing 10% fetal bovine serum (FBS) with or without added bilirubin, 0.01-0.10 mM. Bilirubin caused profound, dose-dependent inhibition of chondrocyte proliferation: after 7 days, control incubations contained 11.45 +/- 2.0 X 10(5) cells per plate versus 4.92 +/- 0.55 X 10(5) cells per plate for wells with added 0.01 mM bilirubin and 1.76 +/- 0.30 X 10(5) for cultures with added 0.05 mM bilirubin. In chondrocytes cultured for 3 days, the addition of 0.05 mM bilirubin was associated with inhibition of [3H]thymidine incorporation into DNA (47 +/- 5% of control), [3H]uridine incorporation into RNA (11 +/- 0.05% of control), and [14C]leucine incorporation into proteins (16 +/- 1% of control). The inhibition of chondrocyte proliferation induced by a range of bilirubin concentrations (0.01-0.10 mM) was in no way attenuated by the addition of physiologic concentrations of albumin (4 g/dl). After 3 days in media containing bilirubin or heme (at equimolar concentrations), chondrocytes were subsequently incubated in FBS alone for an additional 3 days; only partial reversal of the bilirubin (or heme)-induced inhibition was then observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Kernicterus is the end result of injury to the central nervous system by bilirubin and other factors. Serum bilirubin levels alone are poor predictors of kernicterus especially in sick preterm infants. We need a rapid noninvasive indicator of neurotoxicity and impending cell injury. Bilirubin first seems to affect neuronal conduction before energy metabolism is impaired. Unchecked, this may lead to failure of ATP synthesis, resulting in the breakdown of all active intracellular processes. It is now possible to measure energy metabolism in vivo and noninvasively by surface coil 31P NMR spectroscopy. Recent animal studies demonstrate perturbations in energy metabolism when hyperbilirubinemia is associated with an open BBB. Perhaps the further developments in NMR spectroscopy and imaging will permit regional assessment of impending or actual bilirubin neurotoxicity and allow us to identify those infants in need of immediate treatment.

Bilirubin is generally considered to be a diagnostically useful, sometimes toxic, metabolic waste product--and nothing more. Many studies, however, summarized in this article, have shown that bilirubin is an effective lipid-soluble antioxidant in vitro, even at physiologic concentrations, vying with even vitamin E in its ability to intercept and inhibit free radical chain reactions that generate hazardous lipid peroxides. These studies suggest that bilirubin may have a biochemical function in vivo and belong to a group of low-molecular weight antioxidants that together provide protection from cellular damage by endogenous-organic free radicals. Dovetailing with the notion that bilirubin may have a protective function is the recent discovery that heme oxygenase, one of the enzymes responsible for bilirubin formation, is a heat-shock protein, one of a group of proteins that are thought to protect organisms from oxidative and other forms of biochemical stress. Thus, the biochemical path from red to green to yellow may defend as well as degrade, and modest levels of the end product may possibly be physiologically beneficial.

Recent clinical and research findings on kernicterus and bilirubin toxicity are reviewed. Bilirubin binds to cell membranes and appears to interfere with the metabolism, depolarization, and transmitter functions of neurons. The neurobehavioral and clinical findings associated with bilirubin encephalopathy and low bilirubin kernicterus are presented and evaluated. Cardiorespiratory stabilization, avoidance of displacing drugs, and preventive use of phototherapy appear to have decreased the incidence of low bilirubin kernicterus in high-risk newborns.