Influence of Binding on the Toxicity of Bilirubin*

Abstract

The controlled clinical observations of Silverman and coworkers 1 presented the first evidence that drugs bound to albumin could be associated with an increased incidence of bilirubin toxicity. A three-fold increase in the incidence of bilirubin encephalopathy was found at autopsy in premature infants treated prophylactically with sulfisoxazole, and the treated infants had lower serum bilirubins than the control population. Further confirmation was provided by Johnson and associatesZ in jaundiced Gunn rats. They documented an abrupt decrease in the serum bilirubin concentration after injection of anions known to be bound by albumin and found a simultaneous increase in the incidence of bilirubin encephalopathy. By in vitro studies, I reported that addition of protein-bound anions to sera from jaundiced infants was associated with a decrease in the spectral absorption curve of protein-bound bilirubin, and after addition of these anions, some of the serum bilirubin was ultrafilterable. It was therefore proposed 4 that a critical determinant in the toxicity of bilirubin is the concentration of diffusible bilirubin rather than its total extracellular concentration. The bilirubin of interstitial fluid is represented, in FIGURE 1, as a stoichiometric equilibrium between an albumin-bilirubin ligand and unbound bilirubin. It is this latter fraction that, because of its lipid solubility, can readily penetrate plasma cell membranes and exert its toxicity. The presence of anions in the circulation that share a common binding site on albumin with bilirubin or indirectly reduce the affinity of albumin for bilirubin can enhance its cytotoxicity by increasing the concentration of diffusible bilirubin. Measurements of the relative saturation of albumin with bilirubin have permitted the detection of the infant at risk to bilirubin en~ephalopathy.~ These measurements can also provide a means to test drugs that are bound to albumin and thereby might increase the toxicity of bilirubin if used therapeutically in jaundiced newborn infants. The basis for the method we adopted is illustrated in FIGURE 2. Bilirubin, when dissolved in a protein-free solution, has an extinction maximum at 440 nm. An identical bilirubin solution that contains an equimolar amount of albumin exhibits a similar millimolar extinction coefficient, but the maximum is at 460 nm. There is a 14% difference in absorbance at 460 nm between free and protein-bound bilirubin, and therefore addition of a test anion that displaces bilirubin from albumin would be reflected by a decreased absorbance at 460 nm (AOD.,,, ,,,,). By calculating the percentage decrease in absorbance at 460 nm in comparison to an equally diluted control sample, the amount of displacement can be quantitated when corrected for nonbilirubin chromogens. The degree of displacement could theoretically vary between 0 and 100% and would be reflected by a percentage

Antioxidant activity of albumin-bound bilirubin.

“…Although free bilirubin has been shown to interact with purified plasma membranes and microsomes (10), a possible physiological function of bilirubin as a membrane-bound chain-breaking antioxidant has to be questioned because of its toxic properties mentioned above. However, binding of bilirubin to albumin not only sequesters the molecule into a nontoxic form (3,6,10,11) but also distributes the pigment throughout the entire circulation and extravascular space (3,12). Therefore, we tested the antioxidant properties of albumin-bound bilirubin (Alb-BR) toward peroxyl radicals, generated chemically by the thermal decomposition of the water-soluble azo compound 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) (13,14).…”

Bilirubin-Albumin Binding in Neonatal Salicylate Intoxication

“…The apparent lack of interaction between bilirubin and salicylate at the secondary bilirubin binding sites contradicts the interpretation of spectral binding data [45] which has been presented as evidence that the ligands compete at secondary bilirubin binding sites. Shifts in the bilirubin spec trum, however, can be due to bilirubin dimer [46] or other physicochemical changes [47] and do not necessarily indicate competitive second site bind ing.…”

Blood plasma binding of acebutolol and diacetolol in man.

“…Protein-binding is also implicated in the manifestation of certain undesirable side-effects, for example by competitive displacement of albuminbound bilirubin, a drug could increase significantly the total free bilirubin. Many therapeutic agents have been shown to possess the capacity to displace bilirubin and thereby potentiate its toxicity, (Odell, 1973).…”