Unbound Bilirubin Concentration Research Articles

How dangerous Gilbert's syndrome is

Gilbert's syndrome is a benign (functional) hyperbilirubinemia, which is based on a hereditary disorder of bilirubin metabolism, as a result of which the concentration of unbound bilirubin can increase several times. Bilirubin, being a breakdown product of hemoglobin, circulates through the bloodstream, combining with albumin molecules. Such bilirubin is called indirect. In the endoplasmic reticulum, it is conjugated; the enzyme glucuronyltransferase is responsible for this process. In Gilbert's syndrome, as a result of insufficient production of this enzyme, the conjugation process is disrupted, and, as a result, the concentration of unconjugated bilirubin increases. According to statistics, this pathological condition is observed in about 5 % of Russians. This syndrome was first described in 1901 by the French physician Augustin Nicolas Gilbert, and was subsequently named after him. The literature also contains references to this syndrome, described as «constitutional hepatic dysfunction», «familial non-hemolytic hyperbilirubinemia», «idiopathic non-conjugated hyperbilirubinemia». Gilbert's syndrome is inherited in an autosomal recessive manner; men get ill 3–4 times more often than women. A number of scientists associate this with a possible inhibitory effect of testosterone on the enzyme UDP-GT1, which breaks down bilirubin. Clinically, Gilbert's syndrome is manifested by episodes of jaundice caused by an increase in the level of unconjugated bilirubin in the blood serum. Against the background of icterus of the sclera and skin, there is increased fatigue, the appearance of a feeling of bitterness in the mouth, loss of appetite, nausea, and sometimes vomiting. The association of Gilbert's syndrome with functional disorders of the biliary tract, in particular, with gallbladder dyskinesia, is often noted.

Evaluation of unbound bilirubin concentration and total bilirubin/albumin ratio in infants at Gunma Children’s Medical Center

Evaluation of unbound bilirubin concentration and total bilirubin/albumin ratio in infants at Gunma Children’s Medical Center

Influence of bilirubin photoisomers on unbound bilirubin measurement in clinical settings

Measured unbound bilirubin concentration is influenced by bilirubin photoisomers. Bilirubin photoisomers are produced even with only a slight light exposure, and clinical samples are inevitably exposed to light. The objective of the study was to evaluate the influence of bilirubin photoisomers on the measurement of unbound bilirubin using serum of jaundiced neonates during blue light phototherapy. Five neonates treated with phototherapy for hyperbilirubinaemia were enrolled. The samples were taken 12 h after initiation of phototherapy. Samples were processed by irradiation with blue light, by indoor ceiling light, by both blue light and indoor ceiling light or shaded. Bilirubin subfractions, total bilirubin and unbound bilirubin were measured. Compared with the non-irradiated samples, the (EZ)-cyclobilirubin concentration and (ZE)-bilirubin/(ZZ)-bilirubin ratio significantly increased in the blue light-irradiated samples, the (ZE)-bilirubin/(ZZ)-bilirubin ratio significantly increased in the indoor ceiling light-irradiated samples, and the (EZ)-cyclobilirubin, (EZ)-bilirubin and (ZE)-bilirubin/(ZZ)-bilirubin ratio significantly increased in the samples irradiated with both lights. No change was noted in unbound bilirubin in any group. We consider that changes in bilirubin photoisomers induced by light exposure during clinical practice do not influence the measured unbound bilirubin concentration.

Is bilirubin/albumin ratio correlated with unbound bilirubin concentration?

The American Academy of Pediatrics guidelines recommend that the total bilirubin (TB)/albumin (Alb) ratio (B/A ratio), instead of serum concentration of unbound bilirubin (UB), can be used with TB for determining treatment modality for jaundiced newborns ≥ 35 weeks of gestation. It is unknown, however, whether the B/A ratio is actually correlated with serum UB. Four hundred and ninety-seven serum samples were obtained from 209 newborns ≥ 35 weeks of gestation, who were admitted to Kobe University Hospital. Serum UB concentration was measured using the glucose oxidase-peroxidase method. Serum TB and Alb concentrations were measured on spectrophotometry. B/A ratios were calculated and were linearly compared with serum UB. Furthermore, the accuracy of the B/A ratio was evaluated. The B/A ratio was significantly correlated with serum UB concentration. A serum UB concentration of 0.6 µg/dL was in agreement with a B/A ratio of 0.5. For comparison of the number of newborns who had serum UB concentrations ≥ or <0.6 µg/dL and B/A ratios ≥ or <0.5, we found the following characteristics: the concordance rate between serum UB concentrations and the B/A ratio was 94%, sensitivity was 51%, and specificity was 99%. The B/A ratio is significantly correlated with serum UB concentration in newborns ≥ 35 weeks of gestation. The B/A ratio, however, is underestimated when serum UB concentrations are >0.6 µg/dL.

Hypoalbuminemia following Abdominal Surgery Leads to High Serum Unbound Bilirubin Concentrations in Newborns Soon after Birth

Background: The serum concentration of unbound bilirubin (UB), which is bilirubin not bound to albumin (Alb), is a better index than total bilirubin concentration (TB) for identifying infants at risk for developing bilirubin neurotoxicity. The degree to which the hypoalbuminemia following abdominal surgery in jaundiced newborns affects bilirubin binding is unknown. Objective: To determine whether lower Alb occurring in newborns undergoing abdominal surgery shortly after birth results in significantly higher UB in serum versus nonsurgical patients at comparable serum TB. Methods: A matched case-control study was conducted with term and late-preterm newborns. The surgery group included 15 newborns who underwent abdominal operation within 3 days after birth. Clinical and laboratory data (serum UB, TB, and Alb concentrations, UB/TB ratio, and binding constant) in the surgery group were collected and compared with those of 30 control newborns who did not undergo abdominal surgery (control group). Results: Serum UB and the UB/TB ratio in the surgery group were significantly higher than those in the control group (p < 0.02, p < 0.001, respectively), whereas there were no significant differences in serum TB and binding constant between the groups. Serum Alb concentrations in the surgery group were significantly lower than those in the control group (p < 0.001). When pre- and postoperative serum Alb concentrations were compared, there was a significant decrease from 3.4 to 2.7 g/dl (p < 0.001). Conclusions: Our study suggests that hypoalbuminemia following abdominal surgery causes a higher serum UB at comparable serum TB in newborns.

Bilirubin Binding Contributes to the Increase in Total Bilirubin Concentration in Newborns With Jaundice

This study tests the hypothesis that the hourly rate of increase in plasma bilirubin concentration (DeltaBT) would increase significantly with increasing binding avidity. The plasma total bilirubin concentration (B(T)), unbound bilirubin concentration, and albumin concentration values for healthy newborns with jaundice (<or=100 hours of age, >or=35 weeks of gestation, and >or=2.5 kg at birth) were obtained from medical records. DeltaBT (in milligrams per deciliter per hour) was calculated as the slope of BT versus age (in hours). Binding avidity was quantified as the product of the albumin concentration and its bilirubin binding constant (K). Linear correlation was used to test the hypothesis that DeltaBT would increase significantly with K.albumin concentration. The ranges of BT, unbound bilirubin concentration, albumin concentration, and K values for the 21 patients studied were 7.6 to 28.5 mg/dL, 0.53 to 2.52 microg/dL, 2.9 to 4.6 g/dL, and 38 to 163 L/micromol, respectively. DeltaBT correlated significantly with K.albumin concentration (r2=0.23; P=.026). Plasma bilirubin binding avidity contributes significantly to DeltaBT. This component of DeltaBT is associated with a lower risk of bilirubin neurotoxicity, and studies aimed at incorporating plasma bilirubin binding avidity measurements into the algorithms used for management of newborn jaundice seem warranted.

Effect of ibuprofen on bilirubin–albumin binding in vitro at concentrations observed during treatment of patent ductus arteriosus

Background In vitro studies have shown that ibuprofen (IBU) may interfere with bilirubin–albumin binding at concentrations of 100 µg/mL and above. Objectives The present study evaluates the in vitro bilirubin displacement over the range of IBU plasma concentrations observed in vivo during curative treatment of patent ductus arteriosus in preterm infants. Methods Considering that individual plasma concentrations obtained during the clinical development of IBU in preterm infants were ranging between 10 and 70 µg/mL and exceptionally above 100 µg/mL, we used the modified peroxidase method to determine total and unbound bilirubin concentrations without IBU and with IBU over this specific concentration range. Results Total bilirubin and albumin concentrations were respectively 6.6 mg/dL and 2.87 g/dL in pooled newborn plasma. No displacement of bilirubin from its albumin binding sites by IBU was observed over a range of concentrations from 10 to 100 µg/mL. Only a concentration of 200 µg/mL significantly increased the unbound bilirubin by 1.5-fold ( p = 0.0008). Conclusions This in vitro study confirms displacement of bilirubin by a high IBU concentration of 200 µg/mL, however it retrieves no significant displacement over a range of concentrations up to and including 100 µg/mL, i.e. within the range of in vivo concentrations at the recommended dose regimen.

Predicting bilirubin neurotoxicity in jaundiced newborns

The management of jaundice in the newborn infant is an area of clinical practice sorely lacking an evidence-based foundation, and neonatal bilirubin neurotoxicity (kernicterus) continues to occur worldwide. Studies suggest that measuring serum or plasma bilirubin binding, in particular the nonalbumin-bound or unbound bilirubin concentration (Bf), would improve jaundice management as it better predicts bilirubin neurotoxicity than the conventionally used total bilirubin concentration (BT). However, many misconceptions persist regarding the relationships between BT, Bf, the magnitude and distribution of the neonatal bilirubin load, and the risk of bilirubin neurotoxicity. Overcoming these misconceptions and integrating Bf and BT into the management of neonatal jaundice may help move clinical practice from its tradition-based approach centered primarily on BT toward an evidence-based approach that will substantially improve our ability to predict bilirubin neurotoxicity and improve the clinical management of this generally benign, but potentially catastrophic, newborn condition.

The significance of measurement of serum unbound bilirubin concentrations in high‐risk infants

In the management of neonatal hyperbilirubinemia, total bilirubin (TB) concentration is not specific enough to predict the brain damage caused by bilirubin toxicity. Unbound bilirubin (UB) easily passes the blood-brain barrier and causes neurotoxicity. We aimed to evaluate whether serum UB concentration would be a useful predictor of bilirubin encephalopathy in high-risk infants. We measured the serum TB and UB concentrations of 388 newborn infants treated with phototherapy or exchange transfusion for their hyperbilirubinemia at Takatsuki General Hospital between January 2002 and October 2003. Peak serum TB and UB levels and UB/TB ratios were studied on each birthweight group: below 1500 g (very low birthweight), 1500 g-2499 g (low birthweight), above 2500 g (normal birthweight); and several clinical factors influencing hyperbilirubinemia were also studied. Peak serum TB and UB levels increased with increasing birthweight, while UB/TB ratios decreased. The very low birthweight group showed higher UB levels and UB/TB ratios despite lower TB levels in intraventricular hemorrhage or severe infection compared to those in the others. The low birthweight and normal birthweight groups showed higher TB and UB levels in cases of hemolytic disease of the newborn compared to non-hemolytic disease of the newborn cases. Eight of 44 cases showed high UB levels accompanied by abnormal auditory brainstem responses, one of whom subsequently developed ataxic cerebral palsy with hearing loss, whereas the other seven showed transient abnormalities of auditory brainstem responses by the treatment of exchange transfusion or phototherapy. The UB measurement was considered to be significant for the assessment of the risk of bilirubin neurotoxicity and the appropriate intervention for hyperbilirubinemia in high-risk infants.

Patent Ductus Arteriosus: Indomethacin, Ibuprofen, Surgery, or No Treatment at All?

Patent Ductus Arteriosus: Indomethacin, Ibuprofen, Surgery, or No Treatment at All?

Unbound Bilirubin Concentration is Associated With Abnormal Automated Auditory Brainstem Response for Jaundiced Newborns

This study was conducted to determine whether incidental jaundice affects automated auditory brainstem response results. We reviewed the medical charts of jaundiced newborns of > or = 34 weeks of gestation who underwent automated auditory brainstem response testing within 4 hours of plasma total bilirubin concentration and unbound bilirubin concentration measurements. We tested the hypothesis that the likelihood of abnormal automated auditory brainstem response results would increase as total bilirubin and unbound bilirubin concentrations increased. Forty-four infants with proximate total bilirubin concentration, unbound bilirubin concentration, and automated auditory brainstem response measurements were identified, and 4 (9%) had bilateral refer automated auditory brainstem response results. The mean total bilirubin concentration of 21.4 mg/dL (SD: 4.0 mg/dL; range: 14.4-29.5 mg/dL) for the 40 infants with bilateral pass automated auditory brainstem response results was not significantly different from that of 23.0 mg/dL (range: 14.9-33.1 mg/dL) for the 4 infants with bilateral refer automated auditory brainstem response results. However, the mean unbound bilirubin concentration of 1.32 microg/dL (range: 0.22-2.99 microg/dL) for the 40 infants with bilateral pass results was significantly lower than the mean of 2.62 microg/dL (range: 0.88-4.41 microg/dL) for the 4 infants with bilateral refer results. Logistic regression showed that increasing unbound bilirubin concentrations but not increasing total bilirubin concentrations were associated with of bilateral refer automated auditory brainstem response results. The probability of bilateral refer automated auditory brainstem response results increases significantly with increasing unbound bilirubin concentrations but not with increasing total bilirubin concentrations. Because unbound bilirubin concentrations are also more closely correlated with bilirubin neurotoxicity than are total bilirubin concentrations, bilateral refer automated auditory brainstem response results for jaundiced newborns may indicate increased risk of bilirubin neurotoxicity, in addition to the possibility of congenital deafness.

Effect of cucurbitacins on bilirubin–albumin binding in human plasma

The aim of this study is to investigate the effect of three cucurbitacins (Cuc) E, D and I on the bilirubin–albumin binding, both in human serum albumin (HSA) and in plasma. Bilirubin–HSA solution and plasma free of cucurbitacins were prepared as well as others containing serial concentrations of cucurbitacins. The concentration of unbound bilirubin was determined in bilirubin–HSA solution and the direct and total bilirubin concentrations were measured in plasma (with normal or elevated bilirubinemia) by Jendrassik and Grof method. In the conditions we adopted Cuc E and D (to a lesser extent), decreased the levels of unbound bilirubin in bilirubin–HSA solution and decreased direct bilirubin concentration and total bilirubin concentration in plasma in a dose-dependent manner while Cuc I had no effect. The effect of Cuc is related to the presence of native HSA. Thus, when albumin was absent or has been denatured by heating or by urea, Cuc E did not modify bilirubin levels, suggesting that the native structure of albumin is essential for such activity. The interaction of HSA with Cuc E was investigated by fluorescence spectroscopy. Cuc E increased the intrinsic fluorescence of the protein and the magnitude of fluorescence intensity of bilirubin–albumin complex. We concluded that Cuc E and D produced a rearrangement in the structure of albumin, particularly in the domain-II, resulting in an increase in the binding of bilirubin to albumin regardless to whether it's conjugated to glucuronic acid or unconjugated.

Possible Ibuprofen-Induced Kernicterus in a Near-Term Infant with Moderate Hyperbilirubinemia.

A 36-week gestation newborn was admitted to the neonatal intensive care unit for treatment of primary pulmonary hypertension and possible sepsis. The infant developed hyperbilirubinemia on day 4 of life and peaked on day 5 at a total serum bilirubin of 19 mg/dL. Phototherapy was started on day 4 and continued for 5 days. On day 8 of life, ibuprofen was started for fever; a concurrent total serum bilirubin was 15.7 mg/dL. The subsequent hospital course was uneventful, and discharge occurred on day 22 of life. Because the patient failed a hearing screen at discharge, he was referred for a diagnostic audiology workup. He subsequently failed formal audiometric testing on two occasions one week apart, and was given a diagnosis of auditory dys-synchrony and/or auditory neuropathy, consistent with kernicterus. At 5½ months of age, he was reported to be hypotonic and to have frequent arching movements. Since the total serum bilirubin did not exceed 19 mg/dL, concern was raised that ibuprofen may have caused displacement of bilirubin from its albumin binding site, resulting in kernicterus due to excessive unbound bilirubin concentrations. Ibuprofen should be administered with caution in preterm infants at risk for kernicterus.

Bilirubin Unbound: Déjà Vu All Over Again?

During the last century it was widely accepted that when drugs that bind avidly to albumin are displaced from the protein, the resulting increase in the concentration of unbound (free) drug would cause increased biological activity and toxicity.1 Supporting examples were thought to be the severe hypoglycemia that is observed in diabetic patients taking sulfonamides in addition to tolbutamide and the potentiation of the effects of warfarin by phenylbutazone in human volunteers. In both instances, the ancillary drug displaces the therapeutic drug from albumin in vitro. Nowadays, it is recognized that factors other than increased unbound drug were responsible for the observed clinical phenomena, and changes in plasma concentrations of free drug are thought to have little clinical relevance except in unusual situations.1–3 The obvious analogy between bilirubin, with its strong affinity for serum albumin, and highly protein-bound drugs (along with the sulfisoxazole tragedy4) led to suggestions long ago that the concentration of unbound bilirubin in the circulation of jaundiced neonates might be a better predictor of the risk of bilirubin encephalopathy than the total concentration of bilirubin. However, accurate measurement of this minute fraction in clinical samples proved difficult and, according to one authority, beyond the capabilities of instrumentation then available.5 Nevertheless, the notion that unbound-bilirubin measurements might be clinically useful and more discriminating than total bilirubin measurements for identifying infants at most risk of kernicterus has persisted. In this issue of Pediatrics , Wennberg et al6 summarize the reasons for this and re-present the case for investigating whether measurements of unbound bilirubin, based on a peroxidase assay, really are worthwhile in the management of neonatal jaundice. They review the experimental and clinical evidence that the apparent unbound-bilirubin concentration is a better predictor of brain uptake and toxicity of bilirubin than the … Address correspondence to Antony F. McDonagh, PhD, Room S-357, Box 0538, University of California, San Francisco, CA 94143. E-mail: tony.mcdonagh{at}ucsf.edu; or M. Jeffrey Maisels, MB, BCh, Department of Pediatrics, William Beaumont Hospital, 3535 W 13 Mile Rd, 709, Royal Oak, MI 48073. E-mail: jmaisels{at}beaumont.edu

Measurement of unbound bilirubin by the peroxidase test using Zone Fluidics

Background Measuring plasma unbound bilirubin concentration by the peroxidase test is useful in the management of jaundiced newborns. However, the commercially available peroxidase technology is manual, and the unbound bilirubin may be seriously underestimated at the 42-fold sample dilution and single peroxidase concentration used. We investigated improving the test by adapting it to Zone Fluidics, which is a system for automating reactant handling that requires small sample volumes and dilution. Methods A computer-directed Zone Fluidics system was constructed using small diameter tubing to connect in series a water–surfactant reservoir, a bi-directional pump, a multiport selection valve to which peroxidase test reactants (45 μl of sample) are attached with one port open to air, and a spectrophotometer flow cell. Test reactants and air are sequentially aspirated through the valve into the tubing connecting the pump and valve to form a reactant “zone” surrounded by air. The zone is advanced to the spectrophotometer flow cell where total and unbound bilirubin are determined (37 °C) from the absorbance at 460 nm at a 2-fold sample dilution and 4 peroxidase concentrations. Imprecision was assessed in artificial controls and newborn plasma. Plasma results were compared with those obtained using the commercial method. Results The CV for unbound bilirubin in the various controls ranged from 11% to 38% (within day) and 12% to 27% (between days). Triplicate CV measurements for newborn plasma measurements ranged from 0.6% to 31% (mean 11%, n = 47). Mean unbound bilirubin by Zone Fluidics was 5-fold higher than that by the commercial method. Conclusion Zone Fluidics can be used to automate the peroxidase test and overcome many of the limitations of the commercially available peroxidase technology.

Follow-up study of auditory brainstem responses in infants with high unbound bilirubin levels treated with albumin infusion therapy.

Several authors reported that there was a close relationship between unbound bilirubin concentrations and abnormal results of auditory brainstem responses. Full-term infants with high-unbound bilirubin concentrations who were treated with human albumin were followed to evaluate their hearing abilities by using auditory brainstem responses. Fifty-eight infants (gestational age, 39.4 +/- 1.4 weeks; birthweight, 3,245 +/- 435 g) with high unbound bilirubin concentrations (> or = 0.9 micro g/dL) were treated with intensive phototherapy. Twenty infants (control group) received only phototherapy, while 38 others (albumin-treated group) were also given i.v. human albumin administration (1 g/kg bodyweight) during the first 2 h of phototherapy. The follow-up study of auditory brainstem responses was carried out at 6 and 12 months of age. Development quotient tests were carried out at 18 months of age. Abnormalities of auditory brainstem response were detected in three infants in the albumin-treated group and six infants in the control group at 6 months. Two infants in the albumin-treated group and four infants in the control group had improved at 12 months. The results of the follow-up study at 18 months of age in the both groups were normal with development quotient >85. No patients with hearing disability and cerebral palsy were clinically detected at the age of 2 years. The results suggest that albumin priming might be effective for decreasing the rate of auditory brainstem response abnormalities at 6 months.

Auditory Brainstem Response and Unbound Bilirubin in Jaundiced (jj) Gunn Rat Pups

The role of plasma bilirubin-albumin binding in the pathogenesis of kernicterus in human newborns is controversial. Kernicterus in the jaundiced (jj) Gunn rat pup, an animal model for kernicterus, prolongs interwave intervals and decreases wave amplitude in the auditory brainstem response (ABR). Plasma total bilirubin concentration (TBC), albumin concentration, and unbound bilirubin concentration (UBC), a measure of bilirubin-albumin binding, were measured in 16-day-old jj Gunn rat pups (n = 21) and compared with ABR wave latencies, interwave intervals, and wave amplitudes by linear correlation. The UBC, but not the TBC or TBC/albumin ratio, correlated positively and significantly with ABR I–II and I–III interwave intervals (r = 0.55, p = 0.009, and r = 0.60, p = 0.004, respectively). The UBC, but not the TBC or TBC/albumin ratio, predicts bilirubin toxicity, as measured by bilirubin-induced ABR changes in jj Gunn rat pups.

Unbound bilirubin associated with kernicterus: A historical approach

Objective: To determine the unbound bilirubin concentration (UBC) associated with kernicterus with the use of clinical data from clusters of kernicterus after sulfisoxazole and benzyl alcohol administration. Design: Sulfisoxazole at 12 mg/dL and benzoate at 10 mmol/L are associated with kernicterus at total bilirubins near 12 and 10 mg/dL, respectively. The concurrent UBC was estimated by first measuring the drug-induced increases in UBC in plasma and artificial sera (peroxidase-diazo method). The increases were then applied to baseline UBC, determined by linear regression analysis of binding data (peroxidase method) from 86 newborns, at total bilirubins of 12 mg/dL for sulfisoxazole and 10 mg/dL for benzoate. Sensitivity and specificity were determined with existing data. Results: Sulfisoxazole and benzoate increased UBC in artificial sera 2.1-fold and 4.1-fold, respectively, and in plasma (sulfisoxazole) 2.4-fold. Benzoate would increase baseline UBC from 0.29 to 1.19 μg/dL and sulfisoxazole from 0.36 to 0.86 μg/dL. The sensitivity and specificity of a UBC of 0.86 μg/dL for predicting kernicterus are 79% and 92% and for 1.19 μg/dL, 50% and 98%, respectively. Conclusion: Historic data predict that the unbound bilirubin above which kernicterus becomes likely lies between 0.86 and 1.19 μg/dL, in good agreement with existing information. (J Pediatr 2000;137:540-4)

Measurement of Plasma Unbound Unconjugated Bilirubin

A method is described for measuring the unconjugated fraction of the unbound bilirubin concentration in plasma by combining the peroxidase method for determining unbound bilirubin with a diazo method for measuring conjugated and unconjugated bilirubin. The accuracy of the unbound bilirubin determination is improved by decreasing sample dilution, eliminating interference by conjugated bilirubin, monitoring changes in bilirubin concentration using diazo derivatives, and correcting for rate-limiting dissociation of bilirubin from albumin. The unbound unconjugated bilirubin concentration by the combined method in plasma from 20 jaundiced newborns was significantly greater than and poorly correlated with the unbound bilirubin determined by the existing peroxidase method (r = 0.7), possibly due to differences in sample dilution between the methods. The unbound unconjugated bilirubin was an unpredictable fraction of the unbound bilirubin in plasma samples from patients with similar total bilirubin concentrations but varying levels of conjugated bilirubin. A bilirubin-binding competitor was readily detected at a sample dilution typically used for the combined test but not at the dilution used for the existing peroxidase method. The combined method is ideally suited to measuring unbound unconjugated bilirubin in jaundiced human newborns or animal models of kernicterus.

ATP-dependent transport of unconjugated bilirubin by rat liver canalicular plasma membrane vesicles.

The transport of highly purified 3H-labelled unconjugated bilirubin (UCB) was investigated in rat liver plasma membrane vesicles enriched in the canalicular domain and found to be stimulated (more than 5-fold) by the addition of ATP. Other nucleotides, such as AMP, ADP, GTP and a non-hydrolysable ATP analogue (adenosine 5'-[alpha, beta-methylene] triphosphate), did not stimulate [3H]UCB transport, indicating that ATP hydrolysis was necessary for the stimulatory effect. [3H]UCB uptake occurred into an osmotically sensitive space. At an unbound bilirubin concentration ([Bf]) below saturation of the aqueous phase (no more than 70 nM UCB), the ATP-dependent transport followed saturation kinetics with respect to [Bf], with a Km of 26+/-8 nM and a Vmax of 117+/-11 pmol per 15 s per mg of protein. Unlabelled UCB inhibited the uptake of [3H]UCB, indicating that UCB was the transported species. Inhibitors of ATPase activity such as vanadate or diethyl pyrocarbonate decreased the ATP effect (59+/-11% and 100% respectively). Daunomycin, a known substrate for multidrug resistance protein-1, and taurocholate did not inhibit the ATP-dependent [3H]UCB transport, suggesting that neither mdr-1 nor the canalicular bile acid transporter is involved in the canalicular transport of UCB. [3H]UCB uptake (both with and without ATP) in canalicular vesicles obtained from TR- rats was comparable to that in vesicles obtained from Wistar rats, indicating that the canalicular multispecific organic anion transporter, cMOAT, does not account for UCB transport. These results indicate that UCB is transported across the canalicular membrane of the liver cell by an ATP-dependent mechanism involving an as yet unidentified transporter.