ESTIMATION OF BILE PIGMENTS 157 



and could probably be converted into a good quantitative estimation for 

 clinical purposes, provided close attention were paid to the optimal condi- 

 tions for the fluorescence, that the iodine oxidation were carried out with 

 greater care than usual, and that tetrahydromesobilene, not mesobilane, were 

 used as standard. Against the use of iodine for the oxidation of urobilinogen 

 to urobilin, Barrenscheen and Weltmann (17^) raised the objection that 

 bilirubin, which may be present in the urines to be tested, is oxidized by 

 iodine to choletelins which also possess fluorescent zinc salts. No choletelin 

 formation can, however, be observed in urines containing bilirubin under 

 the conditions of the test. 



Rudert and Heilmeyer {2390) have criticized this method and it has fallen 

 into disrepute. There is no doubt that Adler obtained values with it which 

 were far too high, but the cause of this was the use of an impure standard. 

 Then only mesobilane was available, which is easily oxidized, and the oxida- 

 tion of this to mesol)ilene is difficult to carry out quantitatively. Rudert and 

 Heilmeyer also worked with this substance as standard. The urobilinogen 

 of urine and feces, however, consists mainly of tetrahydromesobilane, which 

 is oxidizable without loss to tetrahydromesobilene. The latter can be obtained 

 in pure form and is quite stable. 



Synthetic fluorescent substances, e.g., acridine compounds, have been 

 used for the purposes of comparison {380S7'2,6Jf5,21.5If,237G,2383). So far, 

 they have not been standardized against pure tetrahydromesobilene. 



To ensure that the green fluorescence of the test solution is due to zinc 

 urobilin and not to an acridine compound which may be present in the urine, 

 it is only necessary to acidify the solution. The urobilin fluorescence is 

 destroyed, while that of atebrin, for example, persists. In order to detect 

 urobilin in the presence of atebrin, urobilin is precipitated with basic lead 

 acetate, set free again with oxalic acid, and after neutralization with ammonia, 

 tested with zinc acetate (2893). 



Urobilin can also be determined spectrophotometrically in aqueous or 

 alcoholic solutions containing hydrochloric acid; the varying values obtained 

 by Heilmeyer for the extinction coefficients of urobilins {cf. Tables IX and X) 

 were largely due to the fact that he measured in solutions of the hydrochloride 

 in water or alcohol. The method, however, necessitates extraction of urobilin 

 from the urine, with consequent losses. 



Reduction methods depend on reduction of urobilin to urobilinogen, and 

 the determination of the latter as the red dye obtained by coupling with 

 p-dimethylaminobenzaldchyde. At first reduction by alkaline fermentation 

 was used (4^31), but later Terwen introduced reduction by ferrous hydroxide 

 in alkaline suspension (1735,2750). The condensation with the aldehyde is 

 carried out in ether containing acetic acid, thus avoiding the formation of 

 red indole derivatives (2969). A modification of Terwen's method is used 

 by Watson (2969,298^, cf. also 2521). The dye can be determined colorimet- 

 T\ca\\y (2511)) or spectrophotometrically (1204,1217). Tetrahydromesobilane 

 derived from crystalline tetrahydromesobilene ("stercobilin"), as well as 

 crystalline mesobilane, have been used as standards, giving the same results. 



According to Watson, false positives claimed by Naumann (2012) are of 

 no practical significance since they are only caused by porphobilinogen, 



