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Clinical Chemistry 20: 783-789, 1974;
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Clinical Chemistry, Vol 20, 783-789, Copyright © 1974 by the American Association for Clinical Chemistry

Determination of Unbound Bilirubin in the Serum of Newborns

Jorgen Jacobsen 1 and Richard P. Wennberg 1

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.


Key Words: bilirubin encephalopathy • free and bound bilirubin • binding affinity and capacity • perinatal chemistry • effects of drugs • kernicterus • erythroblastosis • enzymatic assay

Submitted on January 24, 1974
Accepted on May 3, 1974




The following articles in journals at HighWire Press have cited this article:


Home page
 Arch. Dis. Child. Fetal Neonatal Ed.Home page
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]

Home page
 PediatricsHome page
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]

Home page
 CLIN PEDIATRHome page
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]

Home page
 CLIN PEDIATRHome page
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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Copyright © 1974 by the American Association for Clinical Chemistry.