156 IV. BILE PIGMENTS 



biliverdin is a mixture of a blue and yellow pigment, is a step in the wrong 

 direction. Malloy and Evelyn {1852) oxidize the bilirubin with a mixture of 

 hydrogen peroxide, hydrochloric acid, and alcohol, and although this method 

 also does not lead to a uniform oxidation product, it is so far the best oxida- 

 tion method available, since only bilatrienes absorbing in the red are formed 

 (of. 1703). _ 



Biliverdin occasionally accompanies bilirubin in the serum. Methods to 

 estimate it directly are not yet available and the determination from the 

 diflference between total bile pigment as obtained by the oxidation methods 

 and bilirubin as obtained by the van den Bergh reaction can only be con- 

 sidered a rough approximation. 



9.2. Estimation of Urobilin and Urobilinogen 



As ha.s already been stated {cf. Section 6.1.), only part of the 

 urobilin of urine or feces is present as such, the remainder being in the 

 form of its reduced derivative urobilinogen. Furthermore, there are 

 present two different urobilins, together with their corresponding 

 urobilinogens, although this complication is of less importance. 



Since separate determinations of urobilins and urobilinogens {3082) 

 can hardly be recommended, two methods of estimation are available. 

 In the first, the urobilinogens are oxidized to urobilins, which are 

 determined fluorimetrically as the zinc complex, or spectrophoto- 

 metrically in acid solution; in the second, the urobilins are reduced 

 to urobilinogens, which are determined by colorimetry or spectro- 

 photometry of the red dye formed by coupling with p-dimethylamino- 

 benzaldehyde. 



The choice of method depends on a number of factors. Since it is difficult, 

 if not impossible, to extract urobilin itself quantitatively from feces and urine, 

 the reduction methods are preferable for exact physiological experiments, 

 particularly on human feces. Even these methods, however, entail a loss 

 of about i'i^c of the fecal urobilinogen {Jf)S4). On the other hand, determi- 

 nation as urobilin is necessary for some animal feces (rats, rabbits) where 

 reduction by ferrous hydroxide to urobilinogen does not succeed {1G<S<S). 

 Oxidation methods are also well suited to urinary determination, particu- 

 larly for clinical purposes, where extraction of the pigments is unnecessary. 

 For estimations on bile, the oxidation is carried out with ferric chloride, 

 which converts bilirubin present to biliverdin. The latter, is then removed 

 and adsorbed to a ferric hydroxide precipitate, after which the urobilin may 

 be determined in the usual way {lof)72,2383). 



Oxidation methods. Adler {13) oxidized urobilinogen with iodine in alcohol 

 in the presence of zinc acetate and thus obtained the strongly fluorescent 

 urobilin zinc complex, which could be determined fluorimetrically. This 

 method was used as a qualitative test by Schlesinger {2I^J^3). In our opinion 

 it is an excellent semiquantitative test for urobilin {cf. also 2010) in urine. 



