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The neurotoxicity of bilirubin has been extensively studied. Hyperbilirubinemia severe enough to cause kernicterus results in severe neurodevelopmental sequelae in newborn infants(1, 2). Although kernicterus is a rare occurrence in term infants affected by Rh isoimmunization, hyperbilirubinemia is a common occurrence in term and preterm infants. Bilirubin has been shown to be present in the brains of preterm infants in autopsy studies, even in infants who did not have significant elevations in serum bilirubin concentrations(3). Despite advances in the care of newborn infants, bilirubin continues to be a potential cause of morbidity and adverse neurologic sequelae, especially in preterm infants(4). Although it is difficult to distinguish the toxicity of bilirubin from other noxious influences such as hypoxia, ischemia, and prematurity, the effect of bilirubin on the newborn developing brain may contribute to adverse neurologic outcomes.

The NMDA receptor ion/channel complex is contained within neuronal membranes on the synaptic surfaces of neurons(5). It is one of the inotropic glutamate receptors and has an important role during brain development(6). Various subtypes of glutamate receptors have been described, and their structure and function have been reviewed(7, 8). The NMDA receptor is comprised of many different subunits, each containing approximately 1000 amino acids(5, 9). It is a ligand-gated channel that contains multiple recognition sites responsible for modulating its function(10, 11). These include specific sites for glutamate, glycine, and polyamines such as spermine, magnesium, and zinc(12). It also contains a selective ion channel for calcium, sodium, and potassium. MK-801 (dizocilpine) is a noncompetitive antagonist that binds to a site within the channel in its open and activated state.

Excitatory amino acid receptors, such as the NMDA receptor, are important for brain plasticity, neuronal growth, synaptogenesis, and the development of learning, memory, and vision(13). Despite the physiologic role of the NMDA receptor in normal development of the brain, increased activation of the receptor is associated with brain cell injury. The immature brain is more sensitive to overstimulation than the adult brain(13), and the developing animal's brain has an overexpression of NMDA receptors.

NMDA receptors, located throughout the CNS, have high densities in the cerebral cortex and hippocampus(13). NMDA receptor-mediated cortical injury can be decreased by the administration of MK-801, a potent NMDA receptor ion channel blocker(14). Furthermore, hypoxia results in modification of NMDA receptor binding characteristics in the cerebral cortex of the newborn piglet, as shown by decreased number of receptors and higher affinity for [3H]MK-801(15). Because bilirubin is toxic to hippocampal neurons(16), albeit in different sectors, bilirubin-induced neurotoxicity may share common features with hypoxia-induced brain injury by mechanisms mediated by the NMDA receptor.

The present study tests the hypothesis that bilirubin, a compound that binds to the phospholipids in neuronal membranes(17), results in modification of the function of the NMDA receptor/ion channel complex in the cerebral cortex of newborn piglets.

METHODS

Animal preparation. Studies were performed in 13 anesthetized, ventilated, and instrumented newborn piglets, 2-4 d of age. The Institutional Care and Use Committee of the University of Pennsylvania approved the animal care protocols. Anesthesia was induced with 4% halothane and lowered to 1% during surgery while allowing the animals to breathe spontaneously through a face mask. Lidocaine 2% was injected locally before instrumentation for endotracheal tube insertion through a tracheostomy and insertion of aortic and inferior venal caval catheters via femoral vessels. After instrumentation, the use of halothane was discontinued, and anesthesia was maintained with nitrous oxide 79%/oxygen 21% and fentanyl (50 μg/kg) in divided doses throughout the experiment. Tubocurarine (0.3 mg/kg) was administered after connection to a volume ventilator. Arterial blood pH, Pao2, Paco2, pressure, and heart rate were recorded in all animals. The hematocrit was measured. Core body temperature was maintained at 39 °C with a warming blanket. Baseline measurements were obtained after 1 h in both groups after surgery to ensure normal arterial pressures and blood gas values.

After stabilization following surgery, the six piglets assigned to the bilirubin-exposed group received a loading dose of bilirubin (35 mg/kg) over 5-10 min followed by a 4-h continuous bilirubin infusion at a rate of 25 mg/kg/h, similar to previously published protocols(18, 19). Bilirubin purchased from Sigma Chemical Co. (final concentration = 3 mg/mL) was mixed in a buffer solution containing 18.5 volume% 0.1 N NaOH, 44.5 volume% human albumin (5%), and 37 volume% 0.055 M Na2HPO4, with the final pH adjusted to 7.4. The bilirubin:albumin molar ratio = 15:1. The seven piglets in the control group received a similar volume of the buffer solution without bilirubin. Animals in both groups received sulfisoxazole diolamine (Roche Laboratories), 160 ng/kg total dose in three divided doses injected after the 1st, 2nd, and 3rd h of the infusion, to displace the binding of bilirubin to albumin(20). The experiments were performed in a darkened room, and the solutions were wrapped in foil to prevent photodecomposition of the bilirubin solutions. After completion of the bilirubin or buffer infusions, the brains were removed within 5 s, placed in liquid nitrogen, and stored at-80 °C before biochemical analyses. Studies were performed on separate specimens of cortex from each control and bilirubin-exposed brain.

Membrane preparation. The P2 membrane fractions were prepared by a modification of the method described by Williams et al.(21). Brain cortex was homogenized in a 0.32 M sucrose buffer solution containing 10 mM HEPES/1 mM EDTA buffer (pH 7.0). The homogenate was centrifuged at 1,000 × g for 10 min, and the supernatant was centrifuged at 40,000 × g for 1 h. The pellet was resuspended and homogenized in 10 mM HEPES buffer containing 1.0 mM EDTA(pH 7.0) and centrifuged at 40,000 × g for 1 h. The pellet, comprised of P2 membrane fractions, was resuspended, washed, and incubated six times at 32 °C in 10 mM HEPES buffer (pH 7.0) containing 1 mM EDTA. The final pellet was suspended in HEPES/EDTA buffer and stored at -80°C until binding assays were performed. Protein content of the brain cell membrane preparation was determined(22).

[3H]MK-801 and[3H]glutamate binding assays. The[3H]MK-801 binding saturation assay was performed in a concentration range of 0.5 to 50 nM at 32 °C in an assay medium containing 10 mM HEPES(pH 7.0), 80 μg of protein, glycine (100 μM), and glutamate (100 μM). Each sample was analyzed in duplicate. Specific [3H]MK-801 binding was obtained by subtracting nonspecific binding in the presence of 10 μM unlabeled MK-801 from the total binding. After 3 h of incubation, the reaction was stopped by the addition of 8 mL of ice-cold HEPES buffer. The samples were filtered through glass fiber filters and washed with additional buffer. The samples were counted in an LKB Rackbeta 1209 scintillation counter with an efficiency of 65% for 3H.

[3H]Glutamate binding was performed on P2 membrane fractions at 0 °C for 30 min in an assay medium containing 10 mM HEPES buffer (pH 7.0) and 150 μg of protein. [3H]Glutamate was used in increasing concentrations from 25 to 1000 nM. Specific NMDA-displaceable[3H]glutamate binding was obtained in the presence of unlabeled NMDA(100 μM). Nonspecific binding was performed in the presence of unlabeled glutamate (1 mM).

Saturation curves were obtained from the data, and Scatchard plots were constructed for the determination of the Bmax andKd.

Na+,K+-ATPase assay. Hydrolysis of ATP was measured in a 1-mL reaction mixture containing 100 mM NaCl, 20 mM KCl, 3 mM Na-ATP, 3 mM MgCl2, 50 mM Tris-HCl buffer(pH 7.4), and 100 μg of protein. A second reaction mixture was prepared in which KCl was replaced by 1.0 mM ouabain. The samples were preincubated for 5 min at 37 °C. ATP was added to initiate the reaction. The reaction was stopped after 5 min of incubation by the addition of 0.5 mL of 12.5% trichloroacetic acid. The samples were stored on ice for 15 min and centrifuged at 2,000 × g for 10 min at 0-4 °C. Aliquots of the supernatant were taken for the analysis of inorganic phosphate(23). The activity of Na+,K+-ATPase was determined by subtracting the enzyme activity in the presence of ouabain from the total activity. The ouabain-sensitive activity was expressed as micromoles of Pi/mg of protein/h.

Determination of ATP and phosphocreatine. Brain tissue concentrations of ATP and phosphocreatine were determined with a coupled enzyme assay(24). The ATP concentration was calculated from the increase in absorbance at 340 nm for the 20-min period after the addition of hexokinase. Twenty microliters of ADP and 20 μL of creatine kinase were added, and readings were taken at 5-min intervals from zero time until a steady-state was restored. The phosphocreatine concentration was calculated from the increase in absorbance at 340 nm between 0 and 20 min after the addition of creatine kinase.

Determination of serum bilirubin. Whole blood was centrifuged for 5 min, and the plasma was removed. Samples were analyzed in an Ektachem 750 analyzer (Clinical Diagnostics, Inc.).

Statistical analysis. Statistical analysis of biochemical measurements was performed using an unpaired two-tailed t test. All values are expressed as the mean ± SD.

RESULTS

The mean physiologic data for the two groups throughout the experiments were similar (Table 1). After 5 h, however, the mean total bilirubin concentration in the bilirubin-exposed group was significantly higher (p < 0.01). In addition, the mean unconjugated and conjugated bilirubin concentrations in the bilirubin-exposed group were 200.1± 39.3 μmol/L (11.7 ± 2.3 mg/dL) and 242.8 ± 76.9μmol/L (14.2 mg/dL ± 4.5), respectively. An elevated brain tissue concentration of bilirubin in the cerebral cortex and disturbances in brainstem auditory evoked responses have been documented with similar serum bilirubin levels(19).

Table 1 Physiologic data
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Mean tissue concentrations of ATP and phosphocreatine were 33 and 60% lower in the bilirubin-exposed group than control values (p < 0.05), indicating a disturbance in cellular energy metabolism. The activity of Na+,K+-ATPase, an index of neuronal membrane function, was 57.2± 2.2 μmol of Pi/mg of protein/h in the control group and 50.0 ± 6.1 μmol of Pi/mg of protein/h in the bilirubin-exposed group (p < 0.05), a 13% reduction.

Specific [3H]MK-801 binding characteristics are summarized inTable 2. The values for Bmax in the two groups are similar. However, the Kd value was lower(indicating increased affinity of the radioligand for the receptor) in the bilirubin-exposed group (p < 0.001) compared with the value in the control group.Figures 1 and2 show representative Scatchard plots for the two groups.

Table 2 [3H]MK-801 binding data in control and bilirubin-exposed piglets
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Figure 1
figure 1

Representative Scatchard plot of [3H]MK-801 binding data in a control piglet (Bmax = 1.10 pmol/mg of protein, Kd = 6.90 nM, r = 0.88).

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Figure 2
figure 2

Representative Scatchard plot of [3H]MK-801 binding data in a bilirubin-exposed piglet (Bmax = 1.29 pmol/mg of protein, Kd = 4.46 nM, r = 0.98).

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Table 3 summarizes the [3H]glutamate binding characteristics of the NMDA-specific glutamate site in the bilirubin and control groups. The values for Bmax andKd in the two groups, respectively, were not significantly different from each other. Figures 3 and4 show representative Scatchard plots of the two groups.

Table 3 [3H]Glutamate binding data in control and bilirubin-exposed piglets
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Figure 3
figure 3

Representative Scatchard plot of [3H]glutamate binding data in a control (Bmax = 652 fmol/mg of protein,Kd = 154 nM, r = 0.93).

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Figure 4
figure 4

Representative Scatchard plot of [3H]glutamate binding data in a bilirubin-exposed piglet (Bmax = 506 fmol/mg of protein/l nM, Kd = 226 nM, r = 0.86).

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DISCUSSION

There are many adverse effects of hyperbilirubinemia on cellular processes. Both in vitro and in vivo studies have demonstrated disturbances in brainstem auditory evoked potentials, synaptic potentials, oxidative phosphorylation, energy metabolism, and neurotransmitter release and synthesis(18, 25–29). The observation that bilirubin-mediated neurotoxicity is prevented by the administration of MK-801, a potent open-channel blocker of the NMDA receptor, to Gunn rats suggests that the NMDA receptor ion/channel complex may be another mechanism of bilirubin toxicity(30).

In the present study, the reduction in the activity of Na+,K+-ATPase and disturbance in the concentrations of high energy phosphate metabolites confirms the effect of bilirubin on processes linked to cellular membranes(31, 32). The binding of bilirubin, in its unbound form or acid form, to phospholipids located in the plasma membrane may initiate a cascade of events that modifies the NMDA receptor. Aggregates of bilirubin acid that ultimately form may precipitate in the membrane(33) and damage nearby membrane-bound enzymes and receptors, or disrupt the plasma membrane structure so that distant sites on the membrane are modified.

The mechanism of protein phosphorylation that regulates cellular processes(34) may be operative in modification of the NMDA receptor. The activities of protein kinase and phosphatase on protein phosphorylation and dephosphorylation, respectively, in ischemic injury(35) may be important factors in bilirubin-mediated neurotoxicity. It has been shown that bilirubin inhibits the phosphorylation of proteins through a reduction in protein kinase activity in membrane fractions(36), perhaps by noncompetitive mechanisms(37). Modulation of glutamate receptors such as the NMDA ion/channel complex by protein phosphorylation(38) is important for the process of synaptic plasticity(39). The lower Kd value (increased affinity) observed in this study may not be due to alterations in protein phosphorylation mechanisms by bilirubin. The observation in this study that the NMDA-specific glutamate site was not modified suggests that activation of the receptor/ion channel complex may not be directly mediated by bilirubin on the glutamate recognition site. However, if [3H]NMDA (not commercially available at the time of this study) binding assays were performed instead of NMDA-displaceable[3H]glutamate binding studies, the standard deviations stated inTable 3 may have been smaller. As a result, statistical significance might have been achieved. We speculate, however, that bilirubin neurotoxicity may be due to the incorporation of bilirubin molecules within the neuronal membrane, resulting in conformational changes of the ion channel that contains the MK-801 binding site.

The changes in NMDA receptor binding characteristics as shown in this study by an increased affinity (lower Kd) after a bilirubin infusion suggests that the ion channel is in an open and activated state, thus allowing the antagonist MK-801 easier access to its binding site within the channel. Because MK-801 is an open-channel antagonist and can reach its binding site only when the channel is in an open state, MK-801 binding is considered to be an index of NMDA receptor/ion channel complex activation(40). Increased activation of the receptor due to the prolonged infusion of bilirubin may lead to NMDA receptor-mediated excitotoxicity and neuronal injury. Although the changes observed in this study immediately follow an acute exposure to bilirubin, permanent neuronal injury cannot be concluded. Modification of the receptor by bilirubin, however, may potentiate the adverse effects of hypoxia or acidosis on neuronal function. Because the NMDA receptor is vital to many important physiologic functions during brain development, alteration of the receptor may exert subtle effects that may not become apparent until the brain completely develops even in the absence of gross brain damage.

The data in this study show that in vivo exposure to bilirubin increases the affinity of the receptor for [3H]MK-801, indicating bilirubin-induced modification of the receptor in the new-born piglet. We speculate that the strong affinity of neuronal membranes for bilirubin(41) leads to bilirubin-mediated neurotoxicity and results in short- or long-term disruption of neuronal function.