Solubility of Bilirubin in Aqueous Solutions.

Abstract

Recent studies concerning the bilirubinbinding properties of serum proteins ( 1 ) have made it necessary to reexamine the solubility of unconjugated bilirubin in aqueous prnteinfree solutions of varying salt concentrations. In dialysis experiments, hIartin( 2 ) noted that only 0.1 mg % bilirubin was soluble in phosphate buffer of ionic strength 0.1. Hy potentiometric titration, Overbeek cf at?. ( 3 ) found that less than 0.5 mg o/o bilirubin was soluble at pH 7.4 in solutions with an ionic strength ranging from 0.003 to 0.006; but Lucassen (4) encountered difficulties in repeating these determinations unless acetone was added to stabilize the reaction mixture. On the other hand, Odell's studies( 1) appeared to indicate that at pH 7.4. larger amounts of bilirubin may be soluble in proteindfree ultrafiltrates possessing a higher ionic strength. Consequently, the solubility of bilirubin was determined in buffers of varying ionic strengths and pH.Materials and methods. PyrexTAf glassware was used which was prepared as follows: (a) soaked in 0.4N NaOH for 15 minutes: (b) rinsed with distilled water X 5: (c) soaked in 1: 100 solution of 7XfrM for at least 18 hours; (d) rinsed with distilled water ? 20; (e) soaked in 6 s HCl for at least 24 hours; (f) rinsed with distilled water 20; (g) soaked in 3% bovine albumin for 15 min.; (h) rinsed with distilled water x 25.*Aided in part by grants from The Medical Foundation, Inc., and U.S.P.H.S., A-1833. t This work was carried out during the tenure of a Postdoctoral Fellowship from Division of General Med. Sciences, U.S.P.H.S.For centrifugation in the Spinco Model L ultracentrifuge, the LusteroidTnr tubes were prepared as in steps c, d, g, h, and discarded after each experiment. The screw c a p were first treated with SilicladrM and then prepared in the same manner as the Lusteroid tubes .Cold phosphate buffer solutions of 0.01, 0.05, 0.10 and 0.20 mollarity, corresponding at pH 7.4 to an ionic strength of 0.024, 0.120, 0.240 and 0.479 respectively(5) were prepared at various pH values ranging from 7.1 to 7.9. In all subsequent steps the solutions were protected from direct light. Bilirubin (Pfanstiehl) wa5 dissolved in cold sodium hydroxide of appropriate normality to give an approximate pigment concentration of 100 mg o/o. One ml of this freshly prepared sodium bilirubinate solution was added immediately to 10 ml of the corerspnding buffer, mixed by inversion, placed in a LusteraidTM tube and centrifuged for 60 minutes in the dark at room temperature at 30,000 rpm. ( Garg 78,4 10).

Spectrophotometric Characteristics of Bilirubin

“…The solubility of bilirubin in aqueous solution as reported in the literature also varies widely (6,7,19,29). Utilizing a dialysis method, Martin (19) reported that 0.1 mg/100 ml bilirubin was soluble in 0.1 M phosphate buffer at pH 7.4.…”

“…Although the principle has been generally accepted that the height of the molar extinction coefficient of bilirubin in chloroform at 453 nm is an index of purity of the sample (35), widely varying values have been reported for the maximal visible light absorption wave length and molar absorptivity of bilirubin in aqueous solution (10,19,37,38), human serum albumin solution (14,19,32,33,39), and human sera (2,19,26,32). Similarly, reported values for the "solubility" of bilirubin in aqueous solution have varied widely (6,7,19,29). The reasons for this marked variability in maximal absorption wave length and absorptivity from one laboratory to another remain unclear.…”

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

“…The high apparent pK could be explained if the bilirubin acid is removed from t h e equilibrium either by an internal conformational change o r by aggregation and precipitation. Although there is disagreement as t o the exact solubility of bilirubin at pH 7.4 (7, 8 , 35), it appears that bilirubin solutions exceeding 0.1 pM exist in a supersaturated state (6, 7) and become increasingly unstable as the pH decreases t o 7.0 (8,44). The increased affinity between cells and bilirubin with decreasing pH might simply be explained by the increased hydrophobicity of bilirubin and consequent interaction with lipids and lipoproteins in the cell membranes (3 1).…”

Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein, and other anions

“…Unconjugated bilirubin is relatively insoluble in aqueous media at physiologic pH (3,4). Although bilirubin is bound to albumin in plasma (5), only small amounts of albumin are present in liver cells (6), and the rate of transfer of bilirubin into liver is greater than the corresponding rate of transfer of albumin (1).…”