PEDIATRICS Vol. 117 No. 2 February 2006, pp. 523-525 (doi:10.1542/peds.2005-2073)
COMMENTARY |
Bilirubin Unbound: Déjà Vu All Over Again?
a Division of Gastroenterology and the Liver Center, University of California, San Francisco, California
b Department of Pediatrics, William Beaumont Hospital, Royal Oak, Michigan
During the last century it was widely accepted that when drugs that bind avidly to albumin are displaced from the protein, the resulting increase in the concentration of unbound (free) drug would cause increased biological activity and toxicity.1 Supporting examples were thought to be the severe hypoglycemia that is observed in diabetic patients taking sulfonamides in addition to tolbutamide and the potentiation of the effects of warfarin by phenylbutazone in human volunteers. In both instances, the ancillary drug displaces the therapeutic drug from albumin in vitro. Nowadays, it is recognized that factors other than increased unbound drug were responsible for the observed clinical phenomena, and changes in plasma concentrations of free drug are thought to have little clinical relevance except in unusual situations.1–3 The obvious analogy between bilirubin, with its strong affinity for serum albumin, and highly protein-bound drugs (along with the sulfisoxazole tragedy4) led to suggestions long ago that the concentration of unbound bilirubin in the circulation of jaundiced neonates might be a better predictor of the risk of bilirubin encephalopathy than the total concentration of bilirubin. However, accurate measurement of this minute fraction in clinical samples proved difficult and, according to one authority, beyond the capabilities of instrumentation then available.5 Nevertheless, the notion that unbound-bilirubin measurements might be clinically useful and more discriminating than total bilirubin measurements for identifying infants at most risk of kernicterus has persisted. In this issue of Pediatrics, Wennberg et al6 summarize the reasons for this and re-present the case for investigating whether measurements of unbound bilirubin, based on a peroxidase assay, really are worthwhile in the management of neonatal jaundice. They review the experimental and clinical evidence that the apparent unbound-bilirubin concentration is a better predictor of brain uptake and toxicity of bilirubin than the total serum bilirubin concentration, and they recommend a national campaign to measure this fraction and establish normal values for different populations. Déjà vu all over again—or timely suggestion?
Although it is true that the concentration of bilirubin in serum, taken alone, is not a reliable predictor of the risk of bilirubin toxicity, especially in premature and sick low birth weight infants, it is also true that in term and near-term infants, kernicterus is rare unless peak concentrations exceed 25 mg/dL (430 µM), and most reported cases have occurred at levels >30 mg/dL (510 µM).7,8 Systematic application of the recent American Academy of Pediatrics guidelines (for a useful nomogram see www.bilitool.org),9 with prudent early use of phototherapy, a noninvasive, safe, and intrinsically inexpensive treatment, should eliminate most potential cases of bilirubin encephalopathy without the need for additional measurements, albeit at the cost of some overtreatment. The effectiveness of this approach is reflected in the current low need for exchange transfusions even in busy neonatal units.10,11 For example, only 8 of 55128 infants of all birth weights (1.5 of 10000 live births) born between 1988 and 1997 at William Beaumont Hospital required an exchange, and over the same period not a single exchange was performed on 1213 live-born infants who weighed <1500 g at birth. Similarly, from 1992–2002, a combined total of <6 infants per year, on average, received an exchange transfusion at 2 referral perinatal centers in Cleveland, Ohio.11 Newer phototherapy techniques12 and pharmacologic intervention13 may reduce further the need for exchange transfusions. Therefore, if bilirubin concentrations of 25 mg/dL (430 µM) can be prevented, the need for free bilirubin or other predictive measurements on term and near-term infants is probably limited except to reduce redundant treatment.
However, preventing most bilirubin encephalopathy is not good enough, and management of jaundice in preterm and sick very low birth weight infants, for whom the guidelines for phototherapy and exchange transfusion are more empiric and less evidence-based, remains challenging. Although autopsy-proven kernicterus is now rare, a recent study found an association between peak bilirubin concentration and the risk of death, hearing loss, and neurodevelopmental impairment in infants with birth weights of <1000 g.14 For example, Sugama et al15 reported hypotonia and (in 1 infant) choreoathetosis together with classical MRI findings of kernicterus in 2 preterm infants (31 and 34 weeks' gestation) who were not acutely ill and had bilirubin levels of <15 mg/dL (260 µM), and MRI evidence of neurologic bilirubin toxicity was detected in 5 preterm infants (25–29 weeks' gestation), with strikingly low serum albumin levels (1.4–2.1 g/dL) whose peak bilirubin concentrations were only 8.7 to 12 mg/dL (148–203 µM).16 Clearly, an accurate method of identifying such endangered infants would be a boon, and it is in such difficult cases that Wennberg et al6 propose that measurements of free bilirubin would be particularly useful.
Notwithstanding the persuasive arguments presented for this view, free-bilirubin measurements should be viewed circumspectly for several reasons. First, there presently is no method available for measuring free-bilirubin concentrations unambiguously and accurately in clinical samples of plasma or serum. Current peroxidase-based methods may be able to accurately estimate the concentration of free, unbound bilirubin in simple aqueous solutions of bilirubin and albumin,17 but it is uncertain what particular fraction of bilirubin they measure when applied to neonatal samples. Is it all of the bilirubin that is not associated with the high-affinity binding-site on albumin but possibly associated with secondary sites or other plasma constituents, or is it only the free fraction (unbound bilirubin solvated by water)? Second, no automated instruments for measuring this apparent unbound fraction are available commercially, although a semiautomated instrument is available from Japan, and an American prototype instrument was developed recently.25 Third, apparent unbound-bilirubin concentrations vary according to the particular methods used for the peroxidase assay, and only one experimental variant gives absolute values for the concentration.18,19 Standardization, which requires unstable aqueous solutions of bilirubin, is also a potential problem, just as it has been in determinations of total bilirubin concentrations,20 which are orders of magnitude larger than anticipated free-bilirubin concentrations and, in principal, much easier to measure. Fourth, elevations of free bilirubin may be only fleeting and therefore of limited and unreliable diagnostic utility.18,19 Last, hemolysis and the presence of bilirubin conjugates can potentially lead to spurious values.
In addition to these recognized drawbacks, there is one intractable dilemma that has generally been overlooked. An implicit assumption of the peroxidase method is that only a single species of bilirubin is present in blood of infants with unconjugated hyperbilirubinemia. However, this assumption is not valid, as can be readily shown by high-pressure liquid chromatography.21 Blood samples from jaundiced infants invariably contain, in addition to the biosynthetic isomer of bilirubin, at least one other isomer of bilirubin generated by exposure of the patient or the sample to ambient light or phototherapy. Concentrations of the photoisomer, which probably has a lower affinity for albumin and lower toxicity than its parent isomer, will vary but may comprise as much as 10% to 20% of the total bilirubin present and will generally be present at far greater concentrations than that of free bilirubin. If the presence of this photoisomer interferes significantly with measurements of the free concentration of biosynthetic bilirubin, then the peroxidase method is unlikely to be a reliable predictor of which infants are at most risk. If it does not, and there is some evidence that it does not,22 then the specificity of the peroxidase method for the free fraction of native unconjugated bilirubin becomes questionable.
This is not to say that the investigations proposed by Wennberg et al are not worthwhile. If it can be shown that peroxidase measurements really are clinically useful for early identification of infants at most risk of bilirubin toxicity and for preventing unnecessary treatment of others, the method would be of enormous benefit irrespective of what it really measures. After all, direct and indirect bilirubin measurements were diagnostically useful long before it was understood what was being measured. However, before large-scale or multicenter studies are undertaken, improvements in instrumentation and standardization of methodology and a better understanding of the underlying chemistry are necessary. In addition, issues of photoisomer interference,23 sample dilution, and correction for hemoglobin interference need to be definitively dealt with. An alternative and methodologically simpler approach for predicting which infants will be at most risk that also merits reinvestigation is hematofluorometry.24 This promising method, which measures reserve bilirubin-binding capacity by using just a drop or two of blood, never got past the prototype stage but could conceivably be transformed into a clinically useful technique using modern electronic, spectroscopic, and micromechanical instrumentation. Last, in this still poorly understood and confusing field of bilirubin toxicity and kernicterus there is a pressing need for consistent and more scientifically accurate terminology. For example, even the term "free bilirubin" is used in the literature to mean both the fraction of bilirubin not conjugated with glucuronic acid and the fraction not complexed with albumin. Studies of the sort proposed by Wennberg et al might well finally clarify whether peroxidase estimates of free bilirubin really are of clinical diagnostic value. If not, free bilirubin may remain, in the words of songwriter Kris Kristofferson, just another word for nothin' left to lose.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant DK-26307 (to A.F.M.). The order of authorship was decided by coin toss.
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FOOTNOTES |
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Accepted Aug 24, 2005.
Address correspondence to Antony F. McDonagh, PhD, Room S-357, Box 0538, University of California, San Francisco, CA 94143. E-mail: tony.mcdonagh{at}ucsf.edu; or M. Jeffrey Maisels, MB, BCh, Department of Pediatrics, William Beaumont Hospital, 3535 W 13 Mile Rd, 709, Royal Oak, MI 48073. E-mail: jmaisels{at}beaumont.edu
The authors have indicated they have no financial relationships relevant to this article to disclose.
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PEDIATRICS (ISSN 1098-4275). ©2006 by the American Academy of Pediatrics
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