ESTIMATION OF BILE PIGMENTS 155 



alkaline form is unstable (1588) and its use for estimations can therefore not 

 be recommended. The red form (pH 3.0 to 4.0) has an absorption maximum 

 at 530 mju. The absorption curves have been studied by several authors 

 {11 53, 12 16, 1588, 1599, 27 93, 27 9 If). The blue solution in mineral acid has a 

 still stronger maximum at 580 mpt (cmM = 79) {1213, p. 156). 



The red form is more frequently used for the estimation, preferably with 

 suitable buffering. Its color can be compared with that of a cobaltous sul- 

 fate standard, or a methyl red standard buffered to pH 4.63 {1153), or it 

 may be measured spectrophotometrically {1688); with the latter method it 

 is possible to introduce a correction for the slight cloudiness of sera, by 

 measuring at a second wavelength at which the azo dye does not absorb. 

 It is desirable to standardize these solutions with pure bilirubin, which 

 should be freshly recrystallized for this purpose. 



Estimation of bilirubin in the form of the hydrochloride of the azo dye 

 has been suggested by Thannhauser and Andersen {2757) and has been used 

 as a basis of spectrophotometric methods {1216,279i;1213, p. 159). It may 

 be impossible, however, to use this without precipitating serum proteins. 

 Determination of the blue alkaline solution has been used by Jendrassik 

 and Grof {1^1 6) and With {3109). Plasma and serum give the same results 

 {1397,363,3107), but, since it is necessary to avoid hemolysis, serum is 

 preferable. 



Estimation of hiliruhin in the urine. For the estimation of bilirubin in 

 urine, the diazo method has also been used {1022,2522), but absolutely fresh 

 urine must be used to avoid oxidation of bilirubin to biliverdin, which does 

 not couple. The method has also been used after adsorption of bilirubin to 

 a barium sulfate precipitate* and extraction with alkali {10Jfl,1629), but it 

 is doubtful whether this offers any advantages and it may entail losses {2568). 



Methods depending on oxidation to biliverdin. For estimation of bile pig- 

 ments in urine and bile, methods by which bilirubin is oxidized to the blue- 

 green biliverdin in acid solution are more satisfactory, since they include the 

 latter substance in principle. This method was developed as early as 1845 

 by Scherer {2^38) and applied to urine by Huppert {1370). It is the basis 

 of the Fouchet test for abnormal amounts of bile pigment in serum, in which 

 the oxidation is carried out by trichloroacetic acid containing ferric chloride 

 and the biliverdin adsorbed to the protein precipitate. Perchloric acid has 

 also been used {83,^). Several authors {1250,1329,1557,2373,2199,3065) 

 have used the oxidation to biliverdin by the Hammarsten reagent (nitric 

 acid, hydrochloric acid, and alcohol) or by yellow nitric acid for the quantita- 

 tive estimation of bile pigments, and have found higher values particularly 

 in bile, but also in serum, than by the diazo method {1250). 



The green color is compared with that of a standard prepared similarly 

 from bilirubin, or with a copper sulfate-dichromate standard. The basic 

 weakness of these methods is that biliverdin is not the end product of the 

 reaction but is oxidized further to bilipurpurins so that an arbitrary end 

 point is used. This has been correctly stressed by Peterman and Cooley 

 {2139), but their method, which is based on the erroneous assumption that 



* The adsorption of bile pigments on baryta was already known to Berzelius {253). 



