Factors Affecting the Binding of Bilirubin to Serum Albumins: Validation and Application of the Peroxidase Method

Abstract

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...

Kinetics and Specificity of Feline Leukemia Virus Subgroup C Receptor (FLVCR) Export Function and Its Dependence on Hemopexin

“…Unconjugated Bilirubin (UCB) Uptake and Export Studies-NRK/FLVCR and NRK/Neo cells were cultured in 12-well dishes as described above and incubated in working buffer containing 10 M BSA and 10 M highly purified [ 3 H]UCB (specific activity of 10.8 mCi/mmol) biosynthetically labeled in dogs (28,29), corresponding to a free bilirubin concentration of 100 nM (30). After 1, 10, and 30 min, the cells were washed three times with working buffer, and bilirubin uptake was then determined from cell-associated radioactivity.…”

Calculated free bilirubin levels and neurotoxicity

“…2 However, a recent reappraisal has questioned the reliability of such in vitro calculations. 34 Certainly, higher thresholds have been reported in some studies 2 and the toxic B F range in vitro may be broad because of differences in cell type, maturity, function, species of origin and B F exposure conditions across studied lines. 2 The duration of B F exposure is also most likely to be an important variable in determining the nature and extent of bilirubin-induced cell injury in vitro and whether it may be reversible in part or in whole, [52][53][54] as shown with erythrocytes long ago.…”

“…Moreover, the calculated neurotoxic B F levels approximate the threshold serum concentration of unbound bilirubin, measured by a peroxidase method, reported to induce neurological deficits in a teenage patient with Crigler-Najjar type I syndrome, 30 if the reported unbound values are corrected for measurement conditions, namely sample dilution. 3,[31][32][33][34] Measured unbound levels associated with neurological deficits in this patient exceeded 20 nM; actual unbound bilirubin corrected for the B40-fold sample dilution used in the measurement 35 is typically at least 10-fold higher, 31 resulting in a predicted serum B F associated with abnormal neurological signs in this patient of >200 nM. Another report on a teenage patient with Crigler-Najjar syndrome observed the onset of neurological abnormalities at a serum B F of 46.7 nM (B467 nM predicted if corrected for sample dilution) with a return to normal status at a serum B F of 16.1 nM (B161 nM predicted if corrected for sample dilution).…”

“…3,23,[31][32][33][34] Among other factors, k varies as a function of sample dilution, albumin concentration, albumin species and the study medium including the presence of competing compounds. 3,23,[31][32][33][34] In this regard, the very low albumin concentration (3 mM) in the CNS of Gunn rat pups 4 relative to that in serum would be predicted to significantly increase the apparent bilirubin-albumin binding constant k (relative to serum) 33 and result in a lower calculated CNS B F than that reported using serum k values. 4 However, this effect is most likely countered, even offset, by the greatly decreased number of bilirubin-binding sites associated with the markedly lower CNS albumin concentration.…”

Yellowness Is a Threat to Newborn - A Review

“…This is done if the infants have severe anemia, hydrops, or both, even in the absence of high serum bilirubin levels. This is usually done with Rh (−ve) isoimmunization in infants [35]. The numbers of infants are very rare requiring exchange transfusion.…”