Effect of pH on the Interaction of Bilirubin with Albumin and Tissue Culture Cells

Nelson, Thomas L.; Jacobsen, Jørgen; Wennberg, Richard P.

doi:10.1203/00006450-197412000-00010

Cited by 71 publications

(22 citation statements)

References 38 publications

(28 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The B f required for a comparable decrease in viability was more than double that observed with albumin present. Nelson reported similar findings with L929 cells (10). These results can be explained in part by self-aggregation of unbound UCB and by the reservoir function of the albuminbound UCB.…”

supporting

confidence: 75%

Cytotoxicity Is Predicted by Unbound and Not Total Bilirubin Concentration

et al. 2007

Self Cite

View full text Add to dashboard Cite

ABSTRACT:Although it has been suggested that the unbound, free, (B f ) rather than total (B T ) bilirubin level correlates with cell toxicity, direct experimental evidence supporting this conclusion is limited. In addition, previous studies never included a direct measurement of B f , using newer, accurate methods. To test "the free bilirubin hypothesis", in vitro cytotoxicity was assessed in four cell lines exposed to different B f concentrations obtained by varying B T /Albumin ratio, using serum albumins with different binding affinities, and/or displacing unconjugated bilirubin (UCB) from albumin with a sulphonamide. B f was assessed by the modified, minimally diluted peroxidase method. Cytotoxicity varied among cell lines but was invariably related to B f and not B T . Light exposure decreased toxicity parallel to a decrease in B f . In the absence of albumin, no cytotoxicity was found at a B f of 150 nM whereas in the presence of albumin a similar B f resulted in a 40% reduction of viability indicating the importance of total cellular uptake of UCB in eliciting toxic effect. In the presence of albumin-bound UCB, bilirubin-induced cytotoxicity in a given cell line is accurately predicted by B f irrespective of the source and concentration of albumin, or total bilirubin level. (Pediatr Res 62: 576-580, 2007) P lasma levels of unconjugated bilirubin (UCB) are elevated in almost all newborn infants. In some infants with markedly elevated plasma UCB levels, bilirubin causes neurotoxicity, sometimes resulting in permanent neurologic dysfunction (1). Management guidelines for jaundiced term and near-term infants, published by the American Academy of Pediatrics (AAP), are based on the premise that total serum bilirubin concentration (B T ) is the best available predictor of risk for bilirubin-induced neurologic damage (BIND) (2). Clinical evidence has indicated, however, that B T , beyond a threshold value of 20 mg/dL, is a poor discriminator of individual risk for BIND (3,4). Since over 99.9% of total plasma UCB (B T ) is bound to albumin or apolipoprotein D (5) and only unbound bilirubin can enter the brain across an intact blood-brain barrier, the level of unbound "free" bilirubin (B f ) should theoretically provide a more accurate indication of the risk of kernicterus.Published studies of UCB toxicity in cell cultures, conducted with different types of albumin at varied molar ratios of bilirubin/albumin (6 -9) reported an increase in cell damage depending on B T . The hypothesis that cell injury is correlated better with B f was first suggested by Nelson et al. (10) in an in vitro study. Moreover, Ostrow et al. (11) reported a meta analysis of in vitro studies that suggested cytotoxicity was better correlated with B f (11). This conclusion was however based on B f calculations using published binding constants, rather than direct measurement in the culture medium, and was thus limited by unknown variations in binding associated with differences in the composition of the incubation media.In jaundiced newborns...

show abstract

supporting

confidence: 75%

Cytotoxicity Is Predicted by Unbound and Not Total Bilirubin Concentration

et al. 2007

Self Cite

View full text Add to dashboard Cite

show abstract

“…The effect of pH on bilirubin toxicity in vitro was studied quantitatively by Nelson et al (12) using L-929 cell cultures.…”

Section: Discussionmentioning

confidence: 99%

“…The recognition that pH affects each of these reactions differently further complicates this model. Within the physiologic pH range, bilirubin binding to albumin is essentially independent of hydrogen ion concentration (1 1, 12), but bilirubin binding to tissue increases at lower pH (8,(12)(13)(14). Inasmuch as pH influences binding to a variety of cell membranes, mitochondria, phospholipid liposomes, and even sephadex (1 5), the explanation for the effect is more likely to be found in the chemistry of bilirubin than in pH-induced alterations of the membranes.…”

mentioning

confidence: 96%

The Importance of Free Bilirubin Acid Salt in Bilirubin Uptake by Erythrocytes and Mitochondria

Wennberg

1988

Pediatr Res

Self Cite

View full text Add to dashboard Cite

ABSTRACI'. The binding of bilirubin to tissue was studied using adult human erythrocytes and rat liver mitochondria. Tissues were incubated with varying bilirubin-albumin molar ratios, varying albumin concentrations of a given bilirubin-albumin molar ratio, and varying pH. Bilirubin binding by tissue was reversible and stoichiometric with the concentration of the free (nonalbumin bound) bilirubin acid salt (bilirubin monovalent anion). Minimal binding of the bilirubin dianion, the predominant state of bilirubin in plasma, was also suggested. The observations support the "free bilirubin theory" where tissue and albumin compete for binding the body's bilirubin pool. Binding to tissue, however, is not determined by the free bilirubin concentration, but by the concentration of the pH dependent subfraction, the free bilirubin acid salt. Tissue binding and toxicity of bilirubin may result from the surfactant properties of the monovalent anion. (Pediatr Res 23: 443-447, 1988)

show abstract

“…Even fewer studies measured the more meaningful equilibrium concentration of bilirubin during or after incubation (22,28). Ostrow et al (27) recently calculated the B f from molar bilirubin/albumin ratios (B/A) reported in several studies by applying ultrafiltration-determined binding constants for HSA in similar media (15).…”

Section: Bilirubin-albumin Bindingmentioning

confidence: 99%

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

et al. 2006

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Effect of pH on the Interaction of Bilirubin with Albumin and Tissue Culture Cells

Cited by 71 publications

References 38 publications

Cytotoxicity Is Predicted by Unbound and Not Total Bilirubin Concentration

Cytotoxicity Is Predicted by Unbound and Not Total Bilirubin Concentration

The Importance of Free Bilirubin Acid Salt in Bilirubin Uptake by Erythrocytes and Mitochondria

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

Contact Info

Product

Resources

About