Published online April 1, 2008
PEDIATRICS Vol. 121 No. 4 April 2008, pp. 872-873 (doi:10.1542/peds.2007-3643)

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LETTER TO THE EDITOR

Bilirubin-binding Capacity in Premature Infants

Sanjiv B. Amin, MD
Department of Pediatrics
Golisano Children's Hospital at Strong
University of Rochester Medical Center
Rochester, NY 14642

Charles E. Ahlfors, MD
PO Box 2904
Vashon, WA 98070

To the Editor.—

The recent article by Bender et al¹ uses the "bilirubin binding capacity" to assess changes in plasma bilirubin binding associated with gestation and clinical status. They define binding capacity as the total bilirubin concentration (TBC) at which the "high affinity" (clinically important) albumin binding sites are "saturated" with bilirubin. The implication is that the risk of bilirubin neurotoxicity will increase significantly at or beyond this TBC. Because binding capacity is defined as the concentration of bound ligand at infinite ligand concentration,² we feel that the plasma bilirubin binding capacity cannot be reliably obtained from the methodology employed for the following reasons.

The authors obtain the apparent binding capacity of the "high affinity" bilirubin binding site by first titrating the plasma samples with bilirubin and measuring the corresponding increase in unbound (free) bilirubin concentration (B_f). Then, these nonlinear data are combined with the albumin concentrations and subjected to a linear transformation (Scatchard plot), the axis intercepts of which (at least in their samples with "high affinity binding affinities") are interpreted as the "binding capacity" (abscissa) and "binding constant" (ordinate) of the "high affinity" binding site. Unfortunately, this type of analysis applies only when the binding protein (eg, albumin) has specific, noninteractive, and independent binding sites, which is a tenuous assumption at best for bilirubin-albumin binding.³ In addition, problems with bilirubin aggregation and solubility as TBC and B_f increase make it impossible to obtain complete binding isotherms (ie, binding isotherms in which the albumin-bound bilirubin reaches a level that changes little with additional increases in TBC).⁴ Klotz and Hunston² point out that applying Scatchard graphs to incomplete binding isotherms can " ... bend perception to wish and choose an intercept on the r [x] axis [‘capacity’] that corresponds with one's predisposition." Finally, as the authors note, they used only a single peroxidase concentration to measure B_f, which increasingly underestimates the equilibrium B_f as the TBC increases,⁵ and further undermines the interpretation of the axis intercepts of Scatchard plots.

In our opinion, it is more helpful to consider the albumin concentration itself as a practical "bilirubin binding capacity." Albumin concentration decreases as gestational age decreases, meaning that, with all else being equal, the relative risk of bilirubin neurotoxicity at any given TBC increases as gestation decreases. The considerable variation in the bilirubin-albumin binding constant additionally impacts on the relative risk and is best assessed by measuring B_f directly. Because B_f measurements are not currently available in the United States, the TBC/albumin ratio may be used as a reasonable substitute.⁶

REFERENCES

1. Bender GJ, Cashore WJ, Oh W. Ontogeny of bilirubin-binding capacity and the effect of clinical status in premature infants born at less than 1300 grams. Pediatrics. 2007;120 (5):1067 –1073[Abstract/Free Full Text]
2. Klotz IM, Hunston DL. Protein affinities for small molecules: conceptions and misconceptions. Arch Biochem Biophys. 1979;193 (2):314 –328[CrossRef][Web of Science][Medline]
3. Brodersen R. Binding of bilirubin to albumin. CRC Crit Rev Clin Lab Sci. 1980;11 (4):305 –399[Web of Science][Medline]
4. Brodersen R, Funding A, Perdersen AO, Röigaard-Pertersen H. Binding of bilirubin to low-affinity sites of human serum albumin in vitro followed by co-crystallization. Scand J Clin Lab Invest. 1972;29 :443 –446
5. Ahlfors CE, Vreman HJ, Wong RJ, et. al. Effects of sample dilution, peroxidase concentration, and chloride ion on the measurement of unbound bilirubin in premature newborns. Clin Biochem. 2007;40 (3–4):261 –267[CrossRef][Web of Science][Medline]
6. Ahlfors CE. Criteria for exchange transfusion in jaundiced newborns. Pediatrics. 1994;93 (3):488 –494[Abstract/Free Full Text]

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PEDIATRICS (ISSN 1098-4275). ©2008 by the American Academy of Pediatrics

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This Article Extract Full Text (PDF) Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services E-mail this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My File Cabinet Download to citation manager Request Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Amin, S. B. Articles by Ahlfors, C. E. Search for Related Content PubMed PubMed Citation Articles by Amin, S. B. Articles by Ahlfors, C. E. Related Collections Premature & Newborn Social Bookmarking                         What's this?