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STATE-OF-THE-ART REVIEW ARTICLE |
Division of Gastroenterology and the Liver Center, University of California, San Francisco, California
ABSTRACT
Blood from jaundiced neonates often contains several isomers of bilirubin in addition to the biosynthetic isomer that causes kernicterus. These isomers are generated during phototherapy or during normal exposure of infants to ambient light. Their presence is generally overlooked or ignored in clinical measurements of circulating bilirubin concentrations and the interpretation of these values. Whether this is justified or clinically important is presently uncertain. However, the presence of isomers may complicate the accurate measurement of free bilirubin concentrations in blood and the use of such values for identifying jaundiced infants at most risk of kernicterus.
Key Words: bilirubin • laboratory medicine • jaundice • phototherapy • hyperbilirubinemia
C linical neonatologists rely heavily, although by no means exclusively, on bilirubin measurements in the management of infants with neonatal jaundice. Specific bilirubin concentrations can also be crucially important in medicolegal judgements involving bilirubin encephalopathy and kernicterus. In this context, bilirubin refers specifically to the unconjugated biosynthetic and potentially toxic form of the pigment designated rather inscrutably by chemists as 4Z,15Z-bilirubin IX
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Bilirubin concentrations can be estimated by transcutaneous techniques but are more commonly measured by analysis of blood, serum, or plasma. These analyses may involve direct spectrophotometry of serum or lysed whole blood or less-direct methods in which samples are treated with a so-called diazo reagent to convert bilirubin to colored derivatives that then are measured spectrophotometrically. The diazo methods themselves are of 2 types: those performed completely in homogenous solution and those in which reagents are contained in a solid matrix through which the sample and other reagents percolate. Methods are generally standardized by reference to standard bilirubin from the National Bureau of Standards. Oddly enough, the National Bureau of Standards material is not pure but contains, in addition to the natural form of bilirubin, a low proportion of 2 unnatural forms (bilirubin III and bilirubin XIII) that are generated during its isolation from animal bile or gallstones (Fig 1B, inset). Fortunately, having similar properties to bilirubin, these impurities do not seem to lead to significant errors in clinical measurements of unconjugated bilirubin.
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Recently, I was consulted by a chief of neonatology about occasional disconcerting discrepancies in the bilirubin measurements in his unit, which had recently switched from a "wet" diazo method to direct spectrophotometric measurements performed on a blood-gas analyzer because of the faster turnaround time. Before the switch, comparative studies had shown good correlation between the 2 methods, but subsequently, a few puzzling cases had shown up in which discrepancies as high as 90 µM (5 mg%) were seen. Figure 1A shows a high-pressure liquid chromatography analysis of one such sample from an infant who had received exchange transfusion and phototherapy. Below it (Fig 1B), shown for comparison, is a similar analysis of my serum, more senile than neonatal but nevertheless showing essentially just one main yellow peak corresponding to unconjugated bilirubin. In principle, a similar profile with a single, albeit more intense, bilirubin peak is what might be expected for an infant with pure neonatal jaundice uncomplicated by cholestatic disease. What then are the extra peaks (1–4) in the sample from the patient?
The multiple peaks shown in Fig 1A are all different isomeric forms of unconjugated bilirubin.8, 9 That is, they share the same constitution but have different spatial arrangements of the atoms and different properties. The main peak (peak 5) is the natural biosynthetic isomer; the other peaks, which in this case amount in total to 
27% of the total unconjugated bilirubin present, are isomeric bilirubins produced during phototherapy. These isomers can be formed not only during phototherapy but, to a lesser degree, by normal exposure of infants to ambient light (Fig 1C). Brief adventitious exposure of blood samples to light can also result in their formation. Without detailed studies, it is unknown whether the complexity of the bilirubin species in the sample shown in Fig 1A was responsible for the discordant values (182 and 148 µM, respectively) obtained by the blood-gas and diazo methods on the same sample, but it is likely to have played a role.
The potential complexity of the unconjugated bilirubins in the blood of neonates illustrated in Fig 1A has been known for years but is not generally appreciated. By and large it has been ignored or overlooked by analytical instrument manufacturers and in comparative studies of different methods. In the chromatograms shown in Fig 1, the isomers elute in order of their lipophilicity. The normal biosynthetic isomer of bilirubin (Fig 1A, peak 5) is the most lipophilic, whereas the other isomers are relatively hydrophilic. On this (perhaps tenuous) basis, it has been assumed that they are less likely to be taken up into the brain and are potentially less toxic than the biosynthetic isomer. This being so, the bilirubin concentration most important to the clinician is the concentration of the biosynthetic isomer alone (Fig 1A, peak 5). Unfortunately, current clinical methods for measuring bilirubin are incapable of specifically measuring that fraction, and it is unclear to what degree different methods include or exclude the isomers in their total bilirubin estimates. Even high-pressure liquid chromatography, often trumpeted as the gold standard for measuring bilirubin, may miss the presence of isomers if the chromatographic conditions are unsuitable.
Although these arcane details of bilirubinology may be of less than passing interest to the busy clinician, it should be understood that the single number returned by the clinical laboratory may belie the true complexity of the bilirubin species present and may not be an accurate measure of the number that is really needed. Fortunately, most current bilirubin measurements on infants undergoing phototherapy are likely to overestimate, rather than underestimate, the true concentration of the most toxic bilirubin isomer, leading to overtreatment rather than the reverse. In any event, it should be remembered that rapid conversion of bilirubin to the presumably less-toxic isomers will begin as soon as phototherapy begins and long before any decline in the apparent concentration of bilirubin in the circulation.
All but a minute fraction of bilirubin in the circulation is present as a complex with serum albumin. The concentration of this non–albumin-bound fraction has been proposed as a better predictor of the risk of kernicterus than the total bilirubin concentration, a proposal for which there is some experimental support.10 The multiplicity of bilirubin species evident in Fig 1 (A and C) suggests that selective measurement of just the unbound fraction of the unisomerized unconjugated form of bilirubin in some clinical samples might prove difficult.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health grants DK-26307 and DK-26743.
I thank M.J. Daood and Drs T.W.R. Hansen, D.A. Lightner, M.J. Maisels, and J.F. Watchko for helpful comments.
FOOTNOTES
Accepted Mar 29, 2006.
Address correspondence to Antony F. McDonagh, PhD, Division of Gastroenterology, University of California San Francisco, 513 Parnassus Ave, Room S-357, San Francisco, CA 94143-0538. E-mail: tony.mcdonagh{at}ucsf.edu
The author has indicated he has no financial relationships relevant to this article to disclose.
REFERENCES
This article has been cited by other articles:
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  M. J. Maisels and A. F. McDonagh Phototherapy for Neonatal Jaundice N. Engl. J. Med., February 28, 2008; 358(9): 920 - 928. [Full Text] [PDF]
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 R. J. Wong, D. K. Stevenson, C. E. Ahlfors, and H. J. Vreman Neonatal Jaundice: Bilirubin Physiology and Clinical Chemistry NeoReviews, February 1, 2007; 8(2): e58 - e67. [Abstract] [Full Text] [PDF]
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