ExtractSpectrophotometric characteristics of bilirubin at low concentrations (0.005–2.500 mg/100 ml) have been studied under various physical conditions in order to gain a better understanding of the state of bilirubin when preparing “solutions” for laboratory use. Standing, minimal shaking, or stirring of the bilirubin preparations at pH 7.4 progressively reduced and altered the maximal spectral absorption of bilirubin (440 nm) in aqueous buffered media. The shift to 415–420 nm is attributed to oxidation of the pigment whereas shoulder formation is attributed to the formation of large size particles (flocculants). In the presence of antioxidants (L-ascorbic acid and nitrogen gas) and EDTA the maximal absorption peak remained at 440 nm but decreased in magnitude concomitant with development of a progressively increasing shoulder at 480–560 nm. In the absence of antioxidants and EDTA maximal absorption shifted to 415–420 nm and the magnitude of 480–560 nm shoulder formation was less. At the higher concentrations of bilirubin and with reduction in pH of the buffer in the absence of antioxidants, the shift to lower wave lengths was reduced and 480–560 nm shoulder formation was increased. In the absence of antioxidants and EDTA at the lower concentrations of bilirubin and in more alkaline media, the reduction at 440 nm and the shift of maximal absorption to the shorter wave lengths was enhanced. At pH 12, stirring of antioxidant-EDTA-containing solutions of bilirubin resulted in neither a shift of maximal absorption to the shorter wave lengths nor the formation of 480–560 nm shoulder. The formation of 480–560 nm shoulder was accompanied by the visual appearance of turbidity. The formation of flocculants when a “solution” is agitated indicates that significant portions of the pigment were in fact, not in solution and must have existed previously as a finely dispersed colloidal sol or supersaturated solution which progressed to a colloidal sol.Spectral curves of bilirubin, therefore, may represent a composite resulting from four physical states of bilirubin: (1) bilirubin truly in solution with the spectral peak at 440 nm; (2) bilirubin in the fine colloidal dispersion with spectral characteristics similar to those of bilirubin in solution; (3) bilirubin flocculant giving 480–560 nm shoulder; and (4) oxidation products of bilirubin with the spectral peaks lower than 440 nm.Increasing the pH of the aqueous media containing bilirubin (0.05 mg/100 ml) from 7.4 to 12.0 increased the molar extinction coefficient of bilirubin, E4401M, 1cm, progressively to a maximum at pH 12 of 6.35 x 104. Very dilute bilirubin preparations (0.005–0.050 mg/100 ml) in aqueous media, pH 7.4, exhibited spectral evidence of rapid oxidation (more so at higher pH), but spectral shoulder formation was still observed after mechanical agitation. Thus, the solubility of bilirubin in 0.1 M phosphate buffer at pH 7.4 appears to be less than 0.005 mg/ 100 ml.SpeculationUnbound bilirubinin vivo, at concentrations exceeding albumin binding capacity and its aqueous solubility, is believed to exist either as a colloidal sol (micropolymer) or a flocculant (macropolymer). It is proposed that it is in the colloidal sol form (micropolymer) that bilirubin toxicity to brain, kidney, intestinal mucosa, erythrocyte, and other organs initially develops by a process of colloid to surface interaction.