Experimental and clinical data strongly suggest that measurement of Bf in newborns with hyperbilirubinemia will improve risk assessment for neurotoxicity, which emphasizes the need for additional clinical evaluation relating Bf and TSB to acute bilirubin toxicity and long-term outcome. We speculate that establishing risk thresholds for neurotoxicity by using newer methods for measuring Bf in minimally diluted serum samples will improve the sensitivity and specificity of serum indicators for treating hyperbilirubinemia, thus reducing unnecessary aggressive intervention and associated cost and morbidity.

Background: The serum or plasma total bilirubin concentration (BT) has long been the standard clinical laboratory test for evaluating neonatal jaundice, despite studies showing that BT correlates poorly with acute bilirubin encephalopathy (ABE) and its sequelae including death, classical kernicterus, or bilirubin-induced neurological dysfunction (BIND). The poor correlation between BT and ABE is commonly attributed to the confounding effects of comorbidities such as hemolytic diseases, prematurity, asphyxia, or infection. Mounting evidence suggests, however, that BT inherently performs poorly because it is the plasma non–protein-bound (unbound or free) bilirubin concentration (Bf), rather than BT, that is more closely associated with central nervous system bilirubin concentrations and therefore ABE and its sequelae. Content: This article reviews (a) the complex relationship between serum or plasma bilirubin measurements and ABE, (b) the history underlying the limited use of Bf in the clinical setting, (c) the peroxidase method for measuring Bf and technical and other issues involved in adapting the measurement to routine clinical use, (d) clinical experience using Bf in the management of newborn jaundice, and (e) the value of Bf measurements in research investigating bilirubin pathochemistry. Summary: Increasing evidence from clinical studies, clinical experience, and basic research investigating bilirubin neurotoxicity supports efforts to incorporate Bf expeditiously into the routine evaluation of newborn jaundice. .

UB is a more sensitive predictor than either serum bilirubin or B:A MR of abnormal ABR maturation, and hence transient bilirubin encephalopathy in premature newborns with hyperbilirubinemia.

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Objective The objective of this study was to determine if plasma unbound or ‘free’ bilirubin concentration (Bf) measured during the first 30 days of life is associated with subsequent abnormal hearing screening testing by automated auditory brainstem response (AABR) in a diverse population of newborns. Study Design An observational study of newborns receiving AABR, plasma total bilirubin concentration (TBC) and Bf measurements and without underlying conditions known to affect hearing was conducted. Logistic regression was used to determine associations between abnormal AABR and Bf or TBC. The impacts of a variety of clinical factors on the regression model were also assessed. Result A total of 191 patients with birth weights and gestations ranging from 406 to 4727 g and 24 to 42 weeks, respectively, were studied. Among them, 175 (92%) had normal (bilateral PASS) AABR and 16 had abnormal AABR (6 had unilateral REFER AABR, and 10 had bilateral REFER AABR). Mean TBC was not significantly different in babies with normal or abnormal AABR, but mean Bf was greater in the latter group (1.76 versus 0.93 µg per 100 ml, respectively, P = 0.012). Bf, but not TBC, was associated with an abnormal AABR (Bf adjusted odds ratio 3.3, 95% CI 1.8 to 6.1). Comparing receiver-operating characteristics curves, the Bf/TBC ratio was a better predictor of an abnormal AABR than Bf alone. Intraventricular hemorrhage was the only confounding clinical variable. Conclusion An abnormal AABR is associated with an elevated Bf or Bf/TBC ratio, but not the TBC alone. The prevalence of bilirubin neurotoxicity as a cause of audiological dysfunction may be underestimated if the TBC alone is used to assess the severity of newborn jaundice.

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The probability of bilateral refer automated auditory brainstem response results increases significantly with increasing unbound bilirubin concentrations but not with increasing total bilirubin concentrations. Because unbound bilirubin concentrations are also more closely correlated with bilirubin neurotoxicity than are total bilirubin concentrations, bilateral refer automated auditory brainstem response results for jaundiced newborns may indicate increased risk of bilirubin neurotoxicity, in addition to the possibility of congenital deafness.

ABSTRACT:The unbound "free" bilirubin concentration (B f ), not the total bilirubin concentration, is the critical determinant of cellular uptake and toxicity of bilirubin. We compared B f measured by a modified peroxidase method with published data obtained with ultrafiltration and examined conditions that affect the affinity (K F ) of human (HSA) and bovine (BSA) serum albumin for bilirubin. The peroxidase and ultrafiltration methods yielded similar K F values that decreased with increasing HSA concentration and the presence of 50 mM chloride. When related to ionic strength, inhibition of BSAbilirubin binding by chloride, bromide, and sulfate were similar, whereas phosphate buffer had a smaller effect. K F was lower at 37°C than at 25°C for HSA but not for BSA. K F for BSA was similar at pH 7.4 and 8. T he unbound ("free") concentration of unconjugated bilirubin (B f ) in plasma, although less than 0.1% of total bilirubin concentration, is the principal determinant of tissue uptake and toxicity of bilirubin, and plays a critical role in the pathogenesis of bilirubin encephalopathy in jaundiced newborns (1,2) and in patients with Crigler Najjar disease (3). Notwithstanding its biologic significance, B f has rarely been measured in either clinical evaluation of jaundiced newborns or in vitro studies of bilirubin effects and toxicity. This avoidance is due in part to the perceived complexity of B f assays and scepticism regarding their clinical value or accuracy (4 -6).Bilirubin-albumin binding has been studied using a variety of techniques, including fluorescent quenching, bilirubin fluorescence, circular dichroism, Sephadex gel filtration, optical rotary dispersion, dialysis, ultrafiltration, spectrophotometry, and enzymatic oxidation of bilirubin (peroxidase method). Reported association constants for HSA range from 6.7 ϫ 10 6 M Ϫ1 to Ͼ10 8 M Ϫ1 (7-10). The binding constant of HSA has been estimated to be 2-13 times greater than that of BSA (11-13). Considerable variation in serum binding of bilirubin has been reported in newborn infants (2). These differences may be due to direct binding competitors (e.g. sulfonamides), allosteric effects, electrolyte environment, or simply the assay technique, e.g. serum sample dilution (14).The dependency of bilirubin binding affinity on HSA concentration as well as chloride concentration was demonstrated by Weisiger et al. (15) using a complicated procedure in which binding affinity of 14 C-bilirubin was calculated after the sequential removal of labeled impurities by serial ultrafiltration. A more practical technique for measuring B f in clinical or laboratory settings was developed by Jacobsen and Wennberg (16) based on the observation that albumin-binding protects bilirubin from oxidation by HRP. Ahlfors (14,17) has modified the peroxidase method, emphasizing the need to measure B f with two or more HRP concentrations and under the same conditions and albumin concentrations existing in plasma or incubation medium.In this investigation, we replicated and extended the exp...