Abstract
Background: Bilirubin-induced neurologic dysfunction (BIND) is a spectrum of preventable neurological sequelae in jaundiced newborns. Current total plasma bilirubin (BT) concentration thresholds for phototherapy and/or exchange transfusion poorly predict BIND. Methods: The unbound (free) bilirubin (Bf) measured at these BT thresholds provides additional information about the risk for BIND. Bf can be readily adapted to clinical use by determining Bf population parameters at current BT thresholds. These parameters can be established using a plasma bilirubin binding panel (BBP) consisting of BT, Bf, and two empiric constants, the maximum BT (BTmax) and the corresponding equilibrium association bilirubin constant (K). Results: BTmax and K provide the variables needed to accurately estimate Bf at BT < BTmax to obtain Bf at threshold BT in patient samples. Once Bf population parameters are known, the BBP in a newborn can be used to identify poor bilirubin binding (higher Bf at the threshold BT compared with the population) and increased risk of BIND. Conclusion: The BBP can also be used in jaundice screening to better identify the actual BT at which intervention would be prudent. The BBP is used with current BT thresholds to better identify the risk of BIND and whether and when to intervene.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Andersen, D. H., Blanc, W. A., Crozier, D. N. & Silverman, W. A. A difference in mortality rate and incidence of kernicterus among premature infants allotted to two prophylactic antibacterial regimens. Pediatrics 18, 614–625 (1956).
Blanc, W. A. & Johnson, L. Studies on kernicterus; relationship with sulfonamide intoxication, report on kernicterus in rats with glucuronyl transferase deficiency and review of pathogenesis. J. Neuropathol. Exp. Neurol. 18, 165–187 (1959).
Harris, R. C., Lucey, J. F. & Maclean, J. R. Kernicterus in premature infants associated with low concentrations of bilirubin in the plasma. Pediatrics 21, 875–884 (1958).
Hsia, D. Y., Allen, F. H. Jr., Gellis, S. S. & Diamond, L. K. Erythroblastosis fetalis. VIII. Studies of serum bilirubin in relation to Kernicterus. N. Engl. J. Med 247, 668–671 (1952).
Odell, G. B. Studies in kernicterus. I. The protein binding of bilirubin. J. Clin. Invest 38, 823–833 (1959).
Maisels, M. J., Pathak, A., Nelson, N. M., Nathan, D. G. & Smith, C. A. Endogenous production of carbon monoxide in normal and erythroblastotic newborn infants. J. Clin. Invest 50, 1–8 (1971).
Maisels, M. J., Pathak, A. & Nelson, N. M. The effect of exchange transfusion on endogenous carbon monoxide production in erythroblastotic infants. J. Pediatr. 81, 705–709 (1972).
Stevenson, D. K. et al. Prediction of hyperbilirubinemia in near-term and term infants. Pediatrics 108, 31–39 (2001).
Ahlfors, C. E. Unbound bilirubin associated with kernicterus: a historical approach. J. Pediatr. 137, 540–544 (2000).
Ahlfors, C. E. Benzyl alcohol, kernicterus, and unbound bilirubin. J. Pediatr. 139, 317–319 (2001).
Bratlid, D. How bilirubin gets into the brain. Clin. Perinatol. 17, 449–465 (1990).
Davis, D. R. & Yeary, R. A. Effects of sulfadimethoxine on tissue distribution of (14C)bilirubin in the newborn and adult hyperbilirubinemic Gunn rats. Pediatr. Res. 9, 846–850 (1975).
Diamond, I. & Schmid, R. Experimental bilirubin encephalopathy. The mode of entry of bilirubin-14C into the central nervous system. J. Clin. Invest 45, 678–689 (1966).
Oie, S. & Levy, G. Effect of sulfisoxazole on pharmacokinetics of free and plasma protein-bound bilirubin in experimental unconjugated hyperbilirubinemia. J. Pharm. Sci. 68, 6–9 (1979).
Crosse, V. M., Meyer, T. C. & Gerrard, J. W. Kernicterus and prematurity. Arch. Dis. Child 30, 501–508 (1955).
Ahlfors, C. E. & Parker, A. E. Bilirubin binding contributes to the increase in total bilirubin concentration in newborns with jaundice. Pediatrics 126, e639–e643 (2010).
Bender, G. J., Cashore, W. J. & Oh, W. Ontogeny of bilirubin-binding capacity and the effect of clinical status in premature infants born at less than 1300 grams. Pediatrics 120, 1067–1073 (2007).
Lorey, F. W., Ahlfors, C. E., Smith, D. G. & Neel, J. V. Bilirubin binding by variant albumins in Yanomama Indians. Am. J. Hum. Genet 36, 1112–1120 (1984).
Bhutani, V. K., Johnson, L. & Sivieri, E. M. Predictive ability of a predischarge hour-specific serum bilirubin for subsequent significant hyperbilirubinemia in healthy term and near-term newborns. Pediatrics 103, 6–14 (1999).
Ahlfors C. E. The bilirubin binding panel: a Henderson–Hasselbalch approach to neonatal hyperbilirubinemia. Pediatrics 138, e20154378 (2016).
Kuzniewicz, M. W. et al. Incidence, etiology, and outcomes of hazardous hyperbilirubinemia in newborns. Pediatrics 134, 504–509 (2014).
Wickremasinghe, A. C. et al. Risk of sensorineural hearing loss and bilirubin exchange transfusion thresholds. Pediatrics 136, 505–512 (2015).
Wu, Y. W. et al. Risk for cerebral palsy in infants with total serum bilirubin levels at or above the exchange transfusion threshold: a population-based study. JAMA Pediatr. 169, 239–246 (2015).
Hansen, T. W. Acute management of extreme neonatal jaundice—the potential benefits of intensified phototherapy and interruption of enterohepatic bilirubin circulation. Acta Paediatr. 86, 843–846 (1997).
Hansen, T. W. The role of phototherapy in the crash-cart approach to extreme neonatal jaundice. Semin. Perinatol. 35, 171–174 (2011).
Ahlfors, C. E., Amin, S. B. & Parker, A. E. Unbound bilirubin predicts abnormal automated auditory brainstem response in a diverse newborn population. J. Perinatol. 29, 305–309 (2009).
Amin, S. B. et al. Auditory toxicity in late preterm and term neonates with severe jaundice. Dev. Med. Child Neurol. 59, 297–303 (2017).
Amin S. B., et al. Chronic auditory toxicity in late preterm and term infants with significant hyperbilirubinemia. Pediatrics 140, e20164009 (2017).
Cashore, W. J. & Oh, W. Unbound bilirubin and kernicterus in low-birth-weight infants. Pediatrics 69, 481–485 (1982).
Funato, M., Tamai, H., Shimada, S. & Nakamura, H. Vigintiphobia, unbound bilirubin, and auditory brainstem responses. Pediatrics 93, 50–53 (1994).
Ichinomiya, K. et al. Problems with using total serum bilirubin as a criterion for phototherapy in extremely low-birthweight infants. Pediatr. Int. 56, 731–734 (2014).
Morioka, I. et al. Serum unbound bilirubin as a predictor for clinical kernicterus in extremely low birth weight infants at a late age in the neonatal intensive care unit. Brain Dev. 37, 753–757 (2015).
Nakamura, H., Yonetani, M., Uetani, Y., Funato, M. & Lee, Y. Determination of serum unbound bilirubin for prediction of kernicterus in low birthweight infants. Acta Paediatr. Jpn. 34, 642–647 (1992).
Ritter, D. A., Kenny, J. D., Norton, H. J. & Rudolph, A. J. A prospective study of free bilirubin and other risk factors in the development of kernicterus in premature infants. Pediatrics 69, 260–266 (1982).
Shimabuku, R. & Nakamura, H. Total and unbound bilirubin determination using an automated peroxidase micromethod. Kobe J. Med. Sci. 28, 91–104 (1982).
American Academy of Pediatrics. Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics 114, 297–316 (2004).
Maisels, M. J. et al. Hyperbilirubinemia in the newborn infant or =35 weeks’ gestation: an update with clarifications. Pediatrics 124, 1193–1198 (2009).
Maisels, M. J., Watchko, J. F., Bhutani, V. K. & Stevenson, D. K. An approach to the management of hyperbilirubinemia in the preterm infant less than 35 weeks of gestation. J. Perinatol. 32, 660–664 (2012).
Bhutani, V. K. et al. Kernicterus: epidemiological strategies for its prevention through systems-based approaches. J. Perinatol. 24, 650–662 (2004).
Bhutani, V. K., Johnson, L. H. & Shapiro, S. M. Kernicterus in sick and preterm infants (1999-2002): a need for an effective preventive approach. Semin. Perinatol. 28, 319–325 (2004).
Holtzman, N. A. Management of hyperbilirubinemia: quality of evidence and cost. Pediatrics 114, 1086–1088 (2004).
Ip, S. et al. An evidence-based review of important issues concerning neonatal hyperbilirubinemia. Pediatrics 114, e130–e153 (2004).
Johnson, L., Bhutani, V. K., Karp, K., Sivieri, E. M. & Shapiro, S. M. Clinical report from the pilot USA Kernicterus Registry (1992 to 2004). J. Perinatol. 29(Suppl 1), S25–S45 (2009).
Johnson, L. H., Bhutani, V. K. & Brown, A. K. System-based approach to management of neonatal jaundice and prevention of kernicterus. J. Pediatr. 140, 396–403 (2002).
Maisels, M. J. & Newman, T. B. Kernicterus in otherwise healthy, breast-fed term newborns. Pediatrics 96, 730–733 (1995).
Newman, T. B. & Maisels, M. J. Does hyperbilirubinemia damage the brain of healthy full-term infants? Clin. Perinatol. 17, 331–358 (1990).
Newman, T. B. & Maisels, M. J. Evaluation and treatment of jaundice in the term newborn: a kinder, gentler approach. Pediatrics 89, 809–818 (1992).
Watchko, J. F. & Maisels, M. J. The enigma of low bilirubin kernicterus in premature infants: why does it still occur, and is it preventable? Semin. Perinatol. 38, 397–406 (2014).
Newman, T. B. & Klebanoff, M. A. Neonatal hyperbilirubinemia and long-term outcome: another look at the Collaborative Perinatal Project. Pediatrics 92, 651–657 (1993).
Newman, T. B. & Maisels, M. J. Less aggressive treatment of neonatal jaundice and reports of kernicterus: lessons about practice guidelines. Pediatrics 105, 242–245 (2000).
Maisels, M. J. & Newman, T. B. Bilirubin and neurological dysfunction—do we need to change what we are doing? Pediatr. Res. 50, 677–678 (2001).
Atkinson, L. R., Escobar, G. J., Takayama, J. I. & Newman, T. B. Phototherapy use in jaundiced newborns in a large managed care organization: do clinicians adhere to the guideline? Pediatrics 111, e555–e561 (2003).
Chang, P. W., Newman, T. B. & Maisels, M. J. Update on predicting severe hyperbilirubinemia and bilirubin neurotoxicity risks in neonates. Curr. Pediatr. Rev. 13, 181–187 (2017).
Jacobsen, J. & Wennberg, R. P. Determination of unbound bilirubin in the serum of newborns. Clin. Chem. 20, 783 (1974).
Lamola, A. A., Eisinger, J., Blumberg, W. E., Patel, S. C. & Flores, J. Fluorometric study of the partition of bilirubin among blood components: basis for rapid microassays of bilirubin and bilirubin binding capacity in whole blood. Anal. Biochem. 100, 25–42 (1979).
Porter, E. G. & Waters, W. J. A rapid micromethod for measuring the reserve albumin bindingn capacity in serum from newborn infants with hyperbilirubinemia. J. Lab. Clin. Med. 67, 660–668 (1966).
Gitzelmann-Cumarasamy, N. & Kuenzle, C. C. Bilirubin binding tests: living up to expectations? Pediatrics 64, 375–378 (1979).
Levine, R. L. Bilirubin: worked out years ago? Pediatrics 64, 380–385 (1979).
McDonagh, A. F. & Maisels, M. J. Bilirubin unbound: deja vu all over again? Pediatrics 117, 523–525 (2006).
Sunderman, F. W. Jr. Current concepts of “normal values,” “reference values,” and “discrimination values,” in clinical chemistry. Clin. Chem. 21, 1873–1877 (1975).
Lott, J. A., Mitchell, L. C., Moeschberger, M. L. & Sutherland, D. E. Estimation of reference ranges: how many subjects are needed? Clin. Chem. 38, 648–650 (1992).
Honore, B. & Brodersen, R. Albumin binding of anti-inflammatory drugs. Utility of a site-oriented versus a stoichiometric analysis. Mol. Pharmacol. 25, 137–150 (1984).
Klotz, I. M. & Hunston, D. L. Protein affinities for small molecules: conceptions and misconceptions. Arch. Biochem. Biophys. 193, 314–328 (1979).
Huber, A. H. et al. Fluorescence sensor for the quantification of unbound bilirubin concentrations. Clin. Chem. 58, 869–876 (2012).
Iskander, I. et al. Serum bilirubin and bilirubin/albumin ratio as predictors of bilirubin encephalopathy. Pediatrics 134, e1330–e1339 (2014).
Hulzebos, C. V. & Dijk, P. H. Bilirubin-albumin binding, bilirubin/albumin ratios, and free bilirubin levels: where do we stand? Semin. Perinatol. 38, 412–421 (2014).
Hulzebos, C. V. et al. The bilirubin albumin ratio in the management of hyperbilirubinemia in preterm infants to improve neurodevelopmental outcome: a randomized controlled trial—BARTrial. PLoS ONE 9, e99466 (2014).
Lamola, A. A. et al. Neonatal bilirubin binding capacity discerns risk of neurological dysfunction. Pediatr. Res. 77, 334–339 (2015).
Jacobsen, J. Binding of bilirubin to human serum albumin—determination of the dissociation constants. FEBS Lett. 5, 112–114 (1969).
Ahlfors, C. E. et al. Bilirubin binding capacity and bilirubin binding in neonatal plasma. Pediatr. Res. 2718, 5 (2017).
Wennberg, R. P., Rasmussen, L. F., Ahlfors, C. E. & Valaes, T. Mechanized determination of the apparent unbound unconjugated bilirubin concentration in serum. Clin. Chem. 25, 1444–1447 (1979).
Khurshid, F. & Medves, J. Effectiveness of universal hyperbilirubinemia screening on newborn health: a systematic review protocol. JBI Database Syst. Rev. Implement. Rep. 16, 287–290 (2018).
Ahlfors, C. E. et al. Effects of sample dilution, peroxidase concentration, and chloride ion on the measurement of unbound bilirubin in premature newborns. Clin. Biochem 40, 261–267 (2007).
Amin, S. B. Effect of free fatty acids on bilirubin-albumin binding affinity and unbound bilirubin in premature infants. JPEN J. Parenter. Enter. Nutr. 34, 414–420 (2010).
Amin, S. B., Maisels, M. J. & Watchko, J. F. Early lipid infusions and unbound bilirubin in preterm neonates: a cause for concern? J. Pediatr. 184, 6–7 (2017).
Hegyi, T., Kathiravan, S., Stahl, G. E., Huber, A. H. & Kleinfeld, A. Unbound free fatty acids from preterm infants treated with intralipid decouples unbound from total bilirubin potentially making phototherapy ineffective. Neonatology 104, 184–187 (2013).
Ebbesen, F., Foged, N. & Brodersen, R. Reduced albumin binding of MADDS—a measure for bilirubin binding—in sick children. Acta Paediatr. Scand. 75, 550–554 (1986).
Ahlfors, C. E. Effect of serum dilution on apparent unbound bilirubin concentration as measured by the peroxidase method. Clin. Chem. 27, 692–696 (1981).
Ahlfors, C. E., Marshall, G. D., Wolcott, D. K., Olson, D. C. & Van Overmeire, B. Measurement of unbound bilirubin by the peroxidase test using Zone Fluidics. Clin. Chim. Acta 365, 78–85 (2006).
Weisiger, R. A. et al. Affinity of human serum albumin for bilirubin varies with albumin concentration and buffer composition: results of a novel ultrafiltration method. J. Biol. Chem. 276, 29953–29960 (2001).
Acknowledgements
We would like to thank our clinical research coordinators, Martin E. Castillo Cuadrado and Cynthia Montiel, for all their efforts in enrolling subjects and for their technical support. This work was supported, in part, by the Mary L Johnson Research Fund and the Christopher Hess Research Fund.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Ahlfors, C.E., Bhutani, V.K., Wong, R.J. et al. Bilirubin binding in jaundiced newborns: from bench to bedside?. Pediatr Res 84, 494–498 (2018). https://doi.org/10.1038/s41390-018-0010-3
Received:
Revised:
Accepted:
Published:
Version of record:
Issue date:
DOI: https://doi.org/10.1038/s41390-018-0010-3
This article is cited by
-
Integrated machine learning identifies biomarkers for bilirubin-induced Alzheimer’s disease-like lesions in neonates and adults
Scientific Reports (2025)
-
A multi-center evaluation of a device for measurement of bilirubin binding capacity in neonates: the effects of gestational age, Intralipid exposure and illness severity
Journal of Perinatology (2019)
-
Commentary: Bench to bedside–one step closer?
Pediatric Research (2018)


