154 IV. BILE PIGMENTS 



{2757) tried to overcome the difficulty by allowing the' "direct" bili 

 rubin to react before precipitating the serum proteins by alcohol and 

 ammonium sulfate, but even this method does not avoid losses com- 

 pletely {1368) and occasionally fails to give perfectly clear solutions. 

 Methods have therefore been developed which allow coupling 

 without precipitation of serum proteins. They are based either on 

 the principle of Malloy and Evelyn of using alcohol concentrations 

 sufficient to allow coupling but insufficient to precipitate the proteins 

 from diluted serum {1851,2522,637) or on that of Jendrassik and 

 Cleghorn {1^13), who found that caffeine causes "indirect" bilirubin 

 to couple with the diazo reagent. The disadvantage of the Malloy- 

 Evelyn method is that it requires a great dilution of the sera, so that, 

 unless the bilirubin concentration is high, only sensitive photoelectric 

 methods of estimation can be applied. Since there is a distinct 

 danger of development of slight cloudiness by the addition of alcohol, ^ 

 the photoelectric method can give very deceptive results. Thus the 

 method of Jendrassik and numerous modifications of it find increasing 

 application {1022,1J^16,2211,30I^3,3107,3109). 



Estimation of "direct'' and "indirect bilirubin. As will be seen in Chap- 

 ter XI, the differentiation between "direct reacting" and "indirect reacting" 

 bilirubin may be of clinical importance for the distinction of hemolytic 

 jaundice from other types of jaundice, but quantitative differential estima- 

 tions have so far not been shown to be of greater diagnostic value than the 

 simple direct van den Bergh reaction. There is a great deal of arbitrariness 

 attached to definitions of a "direct reaction." Thus Malloy and Evelyn 

 {1851) recommend reading after thirty minutes, Watson {637) after one 

 minute. Definitions of what is meant by a "delayed" or "biphasic" reaction 

 are still more vague. Attempts have been made to determine "direct" 

 bilirubin as the difference between total and "indirect" bihrubin, by extract- 

 ing the latter with chloroform {5^9,2534,2855-2857), but the results thus 

 obtained do not tally with the results by direct estimation of "direct" 

 bilirubin {637). 



Gray and Whidborne {304-3) found a real difference in reaction velocity 

 only between the serum bilirubin in hemolytic jaundice on one hand, and 

 that in obstructive jaundice or hepatitis on the other. With the latter, 

 "prompt," "delayed," and "biphasic" reactions depend solely on bilirubin 

 concentration, the apparent difference in velocity of reaction being caused 

 by the fact that, above a concentration of 1.6 milligram per cent bilirubin 

 (in the final dilution). Beer's law is no longer obeyed by the diazo dye. 

 Van den Bergh's original observation is thus accurate, but confusion has 

 arisen from attempts to elaborate upon the types of reaction. 



Estimation of the dye. The "bilirubin"-azobenzenesulfonic acid dye (c/. 

 Section 4.3.) has indicator properties. It is blue in strongly acid solution, 

 red in weakly acid and neutral solution, and blue in alkaline solution. The 



