FLUORESCENT DYE METHOD FOR DETERMINATION OF BILIRUBIN-BINDING CAPACITY OF SERUM ALBUMIN

Abstract

A simple, rapid microfluorometric method for quantitative measurement of serum albumin binding capacity for unconjugated bilirubin (UB), utilizing a fluorescent dye, Direct Yellow 7, has been developed. Sephadex G-200 column chromatographic study revealed exclusive association of serum albumin fraction with the dye. Spectrofluorometric study demonstrated enhanced fluorescence (ΔF) of the dye when bound to serum albumin. The addition of increasing concentrations of UB (5 to 70 mg%) to adult human sera resulted in a progressive inhibition of ΔF with complete inhibition at 55 mg%. Multiple regression analysis yielded two significantly different slopes (p<.001), first, from 0 to 27 mg% and second slope, 27 to 55 mg%. UB concentration of 27 mg% corresponded to UB: albumin molar ratio of 0.97, while 55 mg% to 1.97. Enhanced fluorescence of the dye resulted entirely from binding of the dye to two bilirubin-binding sites on albumin, since ΔF was completely inhibited by addition of two moles of UB per mole of albumin. Lowered pH reduced albumin binding capacity and exposure to light increased binding capacity of icteric sera. The binding capacity of purified human serum albumin was 7.1±0.8 ΔF/gm albumin (SD) (n=4), approximately one-half that of the adult human sera, 13.4±0.5 ΔF/gm (SD) (n=4), while human cord sera gave 9.3±1.1 ΔF/gm (SD) (n=41), 30% less than that of the adult sera on albumin molar basis.