Mass-spectrometric study of the azopigments obtained from bile pigments with diazotized ethyl anthranilate

Abstract

The structures of some azopigments obtained by diazotization of conjugated and unconjugated bile pigments with diazotized ethyl anthranilate were studied by mass spectrometry. The oco-azopigments derived from rat bile and human bile were shown to be identical (t.l.c. and mass spectra) with azobilirubin derived from unconjugated bilirubin. The presence of two methyl vinyl isomers (Ia) and (Ib) in equal amounts was shown by t.l.c. and mass spectrometry. The structure of the 8-azopigment derived from rat bile was studied by two methods: (a) ammonolysis gave rise to an amide having a CH2 * CH2 * CO * NH2 side chain as shown by its mass spectrum; (b) the mass spectrum of a trimethylsilyl derivative of the 8-azopigment methyl ester confirmed the ester to be a f-D-monoglucuronide ester ofazobilirubin I.

Heterogeneity of bile pigment conjugates as revealed by chromatography of their anthranilate azopigments

“…Diazotization of 1 mol of the tetrapyrrolic bile pigments, bilirubin and conjugated bilirubin, leads to the formation of 2mol of dipyrryl azopigments (Fischer & Haberland, 1935;Overbeek, Vink & Deenstra, 1955;Brodersen, 1960;Lucassen, 1961;Compernolle et al 1970). According to current concepts only bilirubin and bilirubin f-D-diglucuronide are expected to be quantitatively important in bile ofhigher animals and in icteric urine and serum of man (Schmid & Lester, 1966;Lester & Troxler, 1969).…”

“…The identification of the derivatives is based on the following evidence. In all cases the minor derivatives obtained by the ammonia treatment moved as synthetic (Compernolle, Jansen & Heirwegh, 1970).…”

“…with chloroform or with chloroform-methanol (17:3, v/v). The structure of aOM has been studied by mass spectrometry (Compernolle et al 1970 Among the hexuronic acid-containing azopigments only 8-azopigment was hydrolysed by the f-glucuronidase preparations tested. In general no further changes were observed when the incubations were prolonged from 2 to 24h (Table 3).…”

“…In all cases conversion into the corresponding, characteristic derivatives was nearly instantaneous. By mass spectrometry monomethoxycarbonyl ester structures could be assigned to the derivatives obtained from azopigments a0 and 8 (Compernolle et al 1970). …”

Bilirubin conjugates in bile of man and rat in the normal state and in liver disease

“…Dog bile contained relatively large amounts of P-D-xylopyranoside and P-Dglucopyranoside of bilirubin (5,6) whereas the P-D-glucuronoside dominated in bile of rat (4,7). A much more complex composition (4) with existence of disaccharidic conjugating groups (8) was demonstrated for human T-tube bile.…”