ESTIMATION OF BILE PIGMENTS 153 



Violacein, the pigment of Chrornobact. violaceu7n,ma.y be a pyrrole pigment. 

 Tobie (2812) obtained pyrrole bases by reduction of violacein with hydriodic 

 acid. Its constitution is still unknown. Neither its properties {650,2687) nor 

 its composition (1560,3125) indicate any close relationship to biliviolinoid 

 pigments. Wrede gives the composition as C^HssOeNs or C6oH4208N6 and 

 finds that seven to eight moles of hydrogen are taken up on catalytic hydro- 

 genation to the leuco stage. Kogl. however, finds the composition C35H25O6N5 

 or C42H30O7N6, forming a penta-acetate C35Hi806N5(CH3CO)5 or hexa- 

 acetate C42H2207N6(CH3CO)6. Since the substance is insoluble in sodium 

 carbonate, it does not contain carboxylic acid groups. 



9. ESTIMATION OF BILE PIGMENTS 



9.1. Estimation of Bilirubin 



Bilirubin in blood serum is still today ocassionally measured by its light 

 absorption. The "icteric index" of Meulengracht simply compares the color 

 of the serum with that of an 0.01% potassium dichromate solution (1922). 

 Since other yellow pigments occur in the serum (e.g., carotenoids, but also 

 dilute hemoglobin adds to the yellow color) this test can only claim to give 

 a rough picture of the bilirubin level in pathological cases and even then can 

 occasionally be grossly misleading. This is made no better by the use of 

 complicated methods, e.g., spectrophotometry, since hemoglobin and carote- 

 noids, like bilirubin, absorb light in the blue part of the spectrum. Neverthe- 

 less, direct spectrophotometry of bilirubin has often been used, e.g., by 

 Verzar and in the important work of Mann (25^1). This method has been 

 shown by many workers to give high and unreliable values (1216,1223,1997, 

 1998,2138,2869,3107). 



Van den Bergh's method, the method of choice for estimating blood 

 bilirubin, is based on the coupHng with diazotized sulfanilic acid to 

 a dye which is red in weakly acid solution and blue in strongly acid 

 or alkaline solution. The numerous modifications of the original 

 method of van den Bergh {221,23^; cf. many textbooks) which have 

 been suggested and are still being suggested show that an ideal solu- 

 tion has not yet been found. In the original method, the blood pro- 

 teins were precipitated by alcohol and the indirect bilirubin (cf. 

 Section 7.2. and Chapter XI, 8.3.2.) was thus converted to a form 

 reacting with the diazo reagent. The dye was measured colorimet- 

 rically or by comparison with standards without buffering the solution. 



The main disadvantage of this method is the loss of a variable 

 amount of bilirubin (up to 50%) by adsorption on the precipitated 

 proteins. "Direct" bilirubin in particular is liable to be adsorbed. 

 Van den Bergh and Grotepass (227) later suggested precipitation of 

 the protein from alkaline solution. Thannhauser and Andersen 



