Photobilirubin: an early bilirubin photoproduct detected by absorbance difference spectroscopy.

Abstract

Absorption of light converts bilirubin-IXa in solution to a mixture of what are probably cis-trans geometric isomers. This reaction is much faster than other photochemical reactions of bilirubin and reaches photoequilibrium before losses due to photooxidation are significant. At room temperature in the dark in the presence of trifluoroacetic acid or iodine or simply on standing, the photoproducts revert to the natural isomer. They also revert under visible light. Their formation and reversion can be followed by chromatography on polyamide and by absorbance difference spectroscopy.In 1970 Davies and Keohane (1) showed by absorbance difference (AD) spectrophotometry that, upon brief irradiation with visible light, bilirubin-IXa (BR) in chloroform or buffered serum albumin solution is converted rapidly to a photolabile product absorbing at about 490 nm. This transient early photoproduct, which we will call photobilirubint (PBR), was not isolated or identified, but the suggestion has been made that it might play some part in phototherapy of neonatal jaundice (2). Direct evidence to substantiate this has not been published and most subsequent studies on the photochemistry and photometabolism of BR have ignored the reaction.In this paper we wish to confirm and explain the observation of Davies and Keohane and show that PBR formation is an important general photochemical reaction of BR that accompanies or precedes other photochemical transformations of the pigment. Evidence that it is the key reaction in phototherapy of neonatal jaundice will be published elsewhere. MATERIALS AND METHODSBilirubin [Sigma, Koch-Light (Colnbrook, U.K.), or Matheson] was purified as described (3) or by washing a chloroform solution of it three times with 5% aqueous NaHCO3 and crystallization from chloroform/methanol, 1:1 (vol/vol). Purified BR contained less than 5% of IIIa and XIIIa isomer impurities (4) as determined by thin-layer chromatography (TLC) (4) Isolation of PBR was achieved by irradiation of a 0.1 mM solution of BR in 1% methanolic ammonia followed by HPLC separation and collection of the PBR peak. In this reverse-phase system, PBR is eluted before BR. Analytical reagent grade solvents were used as supplied. Reagent grade solvents were purified and redistilled.RESULTS AND DISCUSSION Fig. 1 shows AD spectra generated by irradiation of bilirubin in chloroform and methanol (containing 0.2-1% concentrated ammonium hydroxide) with monochromatic (440 nm, 10-nm bandpass) light. These spectra represent changes in the irradiated sample relative to the unirradiated reference. As reported for chloroform and carbon tetrachloride solutions (1, 2), irradiation produced a "loss" peak near 460 nm and synthesis peaks near 500 and 350 nm. Essentially similar results were obtained with light source C and for solutions of BR in chloroform/1% n-hexane, chloroform/1% ethanol/5-50% triethylamine, chloroform/1% ethanol/40 mM 2,3-dimethyl-2-butene, and benzene. In benzene the 490-nm synthesis peak eventually merged with another broad synth...

pH Dependence of the Water Solubility of Bilirubin Photo-Derivatives and its Relevance to Phototherapy

“…These arise from photoisomerization of bilirubin in the chloroform solutions undergoing irradiation-extraction, as shown by absorption difference spectra and TLC analyses (9). In addition, varying amounts of bilirubin were recovered when aqueous solutions of the photopigment were acidified or kept in the dark at room temperature for a few hours.…”

“…Among them, the three geometric isomers of bilirubin with different configurations about the 5, 15 meso double bond ( Fig. 1) are likely predominant, as supported by chemical and spectroscopic evidence (9,16). Their formation precedes or accompanies other photoreactions of bilirubin, such as photo-oxidation (8), photo-addition of nucleophilic molecules to the 18-vinyl group (4,12,13), photoscrambling to IIIa and XIIa isomers (I), and production of certain poorly characterized yellow products, e.g., photobilirubin I1 (27), "430 pigment" (6), "41 5 pigment" (21), and "peak 1" (5).…”

“…The pigment extracted from chloroform, which we call here "photopigment," for the reason given below, probably includes the water-soluble E-isomers of bilirubin, i.e., the "photobilirubin" described by Lightner et al (9). These arise from photoisomerization of bilirubin in the chloroform solutions undergoing irradiation-extraction, as shown by absorption difference spectra and TLC analyses (9).…”

“…The recovered amounts were checked for identity by TLC (1 7), MS (7) and HPLC (23). Photobilirubin is known to revert easy to bilirubin both thermally and by acid catalysis (9).…”

“…Their formation precedes or accompanies other photoreactions of bilirubin, such as photo-oxidation (8), photo-addition of nucleophilic molecules to the 18-vinyl group (4,12,13), photoscrambling to IIIa and XIIa isomers (I), and production of certain poorly characterized yellow products, e.g., photobilirubin I1 (27), "430 pigment" (6), "41 5 pigment" (21), and "peak 1" (5). The configurational photoisomers of bilirubin, commonly known as photobilirubin (9), appear to be more polar, less lipophilic, and probably more acidic than the natural pigment (10). These properties are a consequence of the reduction in the number of intramolecular hydrogen bonds involving the carboxy groups of bilirubin (14) (Fig.…”