Cytotoxicity Is Predicted by Unbound and Not Total Bilirubin Concentration

Abstract

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...

Bilirubin accumulation and Cyp mRNA expression in selected brain regions of jaundiced Gunn rat pups

“…This process becomes clinically relevant when the load of bilirubin is excessive or when agents competitively displace UCB from binding sites on albumin (3,5). Because of multiple factors affecting UCB binding and Bf, it has not been possible to define a bilirubin/albumin molar ratio (B/A) considered to be safe in protecting against BIND.…”

“…Recent findings emphasize that diffusion of unconjugated bilirubin (UCB) into cells, causing neurotoxicity, is related to the unbound, free fraction of plasma UCB (Bf), rather than the total plasma UCB level (3)(4)(5).…”

Unbound (Free) Bilirubin: Improving the Paradigm for Evaluating Neonatal Jaundice

“…It has been now been demonstrated directly that in vitro cytotoxicity is accurately predicted by B f but not B T , irrespective of whether human or bovine serum albumin or fetal calf serum is used in the medium (59 ). Such measurements with cultured neurons and astrocytes may provide considerable insight into the concentrations of B f in the cerebrospinal fluid (CSF) and extracellular fluid of the CNS that are likely to produce bilirubin neurotoxicity in vivo (73 ). In addition, B f measurements may help evaluate the considerable variation in susceptibility of cell lines to bilirubin toxicity and the specific anatomical patterns of kernicterus that occur in vivo (74 ).…”

Effect of storage and freezing on unbound bilirubin measurement

“…Emerging evidence suggests that the fraction of TSB not bound to albumin, referred to as the unbound bilirubin concentration (B f ), may be a better predictor of bilirubin-induced neurotoxicity than TSB. [1][2][3] A semi-automated, Food and Drug Administration approved method based on the peroxidase method has been most widely used to measure B f [4][5][6] but is not routinely available in clinical laboratories. [7] Current bilirubin research is aimed at evaluating the usefulness of B f .…”