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Clinical Chemistry, Vol 20, 783-789, Copyright © 1974 by the American Association for Clinical Chemistry
1 Department of Pediatrics, Division of Neonatal Biology, University of Washington, Seattle, Wash. 98195.
An enzymatic assay is described for non-albuminbound bilirubin in the serum of newborn infants. Unbound bilirubin is oxidized to colorless compounds by ethyl hydroperoxide in the presence of horseradish peroxidase (EC 1.11.1.7), while albumin-bound bilirubin is protected from oxidation. Because the equilibrium between albumin and bilirubin occurs rapidly, the oxidation step is rate limiting, and the initial oxidation velocity of total bilirubin is proportional to the unbound bilirubin concentration. By titrating serum with bilirubin in vitro, the association constant and binding capacity of high-affinity sites for albumin binding can be determined. Normal human serum albumin tightly binds 1 mole of bilirubin per mole of albumin (binding constant, 2-4 x 108 liter/mol). Although weaker secondary binding occurs, the unbound bilirubin fraction increases rapidly after the high-affinity binding sites are saturated. Compromised newborns may have a decreased apparent binding capacity and (or) binding affinity. The method can be used to assess the risk of a jaundiced infant for bilirubin encephalopathy.
Submitted on January 24, 1974
Accepted on May 3, 1974
The following articles in journals at HighWire Press have cited this article:
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  S. B. Amin, T. Harte, L. Scholer, and H. Wang Intravenous Lipid and Bilirubin-Albumin Binding Variables in Premature Infants Pediatrics, July 1, 2009; 124(1): 211 - 217. [Abstract] [Full Text] [PDF]
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 C. E. Ahlfors, R. P. Wennberg, J. D. Ostrow, and C. Tiribelli Unbound (Free) Bilirubin: Improving the Paradigm for Evaluating Neonatal Jaundice Clin. Chem., July 1, 2009; 55(7): 1288 - 1299. [Abstract] [Full Text] [PDF]
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A. F. McDonagh, H. J. Vreman, R. J. Wong, and D. K. Stevenson Photoisomers: Obfuscating Factors in Clinical Peroxidase Measurements of Unbound Bilirubin? Pediatrics, January 1, 2009; 123(1): 67 - 76. [Abstract] [Full Text] [PDF]
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 C V Hulzebos, D E van Imhoff, A F Bos, C E Ahlfors, H J Verkade, and P H Dijk Usefulness of the bilirubin/albumin ratio for predicting bilirubin-induced neurotoxicity in premature infants Arch. Dis. Child. Fetal Neonatal Ed., September 1, 2008; 93(5): F384 - F388. [Abstract] [Full Text] [PDF]
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J. C. Sinclair A Difference in Mortality Rate and Incidence of Kernicterus Among Premature Infants Allotted to Two Prophylactic Antibacterial Regimens, by William A. Silverman, et al, Pediatrics, 1956;18:614-624 Pediatrics, July 1, 1998; 102(1): 225 - 227. [Abstract] [Full Text]
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 T. W. R. Hansen Bilirubin in the Brain: Distribution and Effects on Neurophysiological and Neurochemical Processes Clinical Pediatrics, August 1, 1994; 33(8): 452 - 459. [PDF]
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 M. Rubin, D. Harell, N. Naor, A. Moser, E. Wielunsky, P. Merlob, and D. Lichtenberg Lipid Infusion With Different Triglyceride Cores (Long-Chain vs Medium-Chain/Long-Chain Triglycerides): Effect on Plasma Lipids and Bilirubin Binding in Premature Infants JPEN J Parenter Enteral Nutr, November 1, 1991; 15(6): 642 - 646. [Abstract] [PDF]
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A. F. Robertson, W. B. Karp, H. C. Davis, W. Zack Catterton, and C. Bunyapen Predicting the Need for Exchange Transfusion in Newborn Infants: A Comparison of Five Methods Clinical Pediatrics, August 1, 1983; 22(8): 533 - 536. [Abstract] [PDF]
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 A. F. Robertson, W. B. Karp, C. Bunyapen, W. Z. Catterton, and H. C. Davis Clinical and Chemical Correlates of the Bilirubin-Binding Capacity in Newborns Arch Pediatr Adolesc Med, June 1, 1981; 135(6): 525 - 528. [Abstract] [PDF]
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