Separation by thin-layer chromatography and structure elucidation of bilirubin conjugates isolated from dog bile

Abstract

1. A system for separation of bile pigments by t.l.c. and for their structure elucidation is presented. Separated bile pigments are characterized by t.l.c. of derived dipyrrolic azopigments. 2. At the tetrapyrrolic stage hydrolysis in strongly alkaline medium followed by t.l.c. demonstrates the presence of bilirubin-IIIalpha, -IXalpha and -XIIIalpha and allows assessment of their relative amounts. 3. Most structural information is derived from analysis of dipyrrolic azopigments. Such derivatives, obtained by treatment of separated bile pigments with diazotized ethyl anthranilate, were separated and purified by t.l.c. Micro methods showed (a) the nature of the dipyrrolic aglycone, (b) the nature of the bonds connecting aglycone to a conjugating group, (c) the ratio of vinyl/isovinyl isomers present in the aglycone and, (d) the nature of the conjugating groups (by suitable derivative formation and t.l.c. with reference to known compounds). 4. In bile of normal dogs at least 20 tetrapyrrolic, diazo-positive bile pigments could be recognized. Except for two pigments the tetrapyrrolic nucleus corresponded predominantly to bilirubin-IXalpha. All conjugated pigments had their conjugating groups connected in ester linkage to the tetrapyrrolic aglycone, Apart from bilirubin-IXalpha, monoconjugates and homogeneous and mixed diconjugates of bilirubin were demonstrated; conjugating groups of major importance were xylose, glucose and glucuronic acid. 5. Bilirubin isomer determination on native bile and isolated bile pigments, and dipyrrole-exchange assays with [14C8]bilirubin indicated (a) that the conjugates pre-exist in bile, and (b) that no significant dipyrrole exchange occurs during isolation of the pigments.

The isolation and further characterization of the bilirubin tetrapyrroles in bile-containing human duodenal juice and dog gall-bladder bile

“…Heirwegh et al (1975) have reported the resolution of 20 different bilirubin tetrapyrroles from dog gall-bladder bile in a t.l.c. system.…”

“…Although she was not able specifically to isolate and define the various pigment species, her results suggested that dog bile contains a relatively greater proportion of glucose-containing diconjugates. Heirwegh et al (1975), in contrast, isolated and characterized more than 20 different bilirubin tetrapyrrole conjugates from dog bile. The procedure that they introduced is difficult to work with, and some of the chemical species encountered appear to have been created in their isolation procedures.…”

Excretion of Conjugated Bilirubin in the Isolated Perfused Rat Kidney

“…Techniques for the isolation of these glucuronide conjugates from bile have been developed, but at the present time they are only appropriate for the production of trace amounts (Ostrow & Murphy, 1970;Wolkoff, Scharschmidt, Plotz & Berk, 1976) and do not give complete separation of the two types of glucuron ide, although this can be improved by chromatography (Jacobsen, 1969;Thompson & Hofmann, 1971;Heirwegh et al, 1975;Gordon et al, 1976). In this study it was necessary to use a method which gave high yields of pigment and hence the procedure described by Lucassen (1961) was employed.…”

“…It is now generally accepted that bilirubin diglucuronide is the major pigment in human and rat bile (Heirwegh et al, 1975;Gordon et ai, 1976); small quantities of bilirubin monoglucuronide are also present (Ostrow & Murphy, 1970;Jansen & Billing, 1971). Techniques for the isolation of these glucuronide conjugates from bile have been developed, but at the present time they are only appropriate for the production of trace amounts (Ostrow & Murphy, 1970;Wolkoff, Scharschmidt, Plotz & Berk, 1976) and do not give complete separation of the two types of glucuron ide, although this can be improved by chromatography (Jacobsen, 1969;Thompson & Hofmann, 1971;Heirwegh et al, 1975;Gordon et al, 1976).…”

“…In order to simplify interpretation of the complex azopigment chromatograms (Heirwegh, Fevery, Michiels, Van Hees & Compernolle, 1975) the results were calculated as the proportions of a 0 (azodipyrrole) and <5 (/?-D-monoglucuronoside), with the contribution from all other diazo-positive bands, namely β + y, being added together (R). The findings have been expressed in this manner since the % of monoconjugates may be equated with twice the value of a a and azopigment δ indicates the proportion of diglucuronide (Gordon, Goresky, Chang & Perlin, 1976).…”