808 



however, be admitted that neither tliis nor any other colour-reaction 

 enables us to identify the presence of bilirubin with absohite certainty. 

 Tlie possibiHty of other substances contained in the serum giving 

 the same reaction with the diazo-body may be esteemed less probable, 

 it cannot with absolute certainty be denied. 



As far as we know it has hitherto been found impossible to 

 isolate bilirubin from normal human serum, which would have 

 afforded an incontestable proof of its presence. Hammersten has 

 attempted it ^). But though in a great majority of cases he obtained 

 tine bilirubin-crystals from horse-serum, lie never succeeded in ob- 

 taining them from normal human serum. From the terms used in 

 the latest edition of his Handbook of Physiological Chemistry we 

 gather that this investigator is not quite convinced yet of the presence 

 of biluribin in normal human serum. 



With a view to researches on anhepatic bilirubin-formation we 

 needed a method which would enable us to identify with absolute 

 certainty the presence of small quantities of bilirubin in bloodserum, 

 exsudates and transsudates, if possible by obtaining the pigment in 

 the form of crystals. After some experiments we have succeeded in 

 this, starting from the property of bilirubin — which we have not 

 found mentioned anywhere — of dissolving readily in acetone. 



To 10 cm', of bloodserum 20 cm', of pure colourless acetone are 

 added. An albumen precipitate is formed, which is centrifugalized. 

 The fluid at the top, coloured more or less intensely yellow, contains 

 all the bilirubin and only traces of albumen. This liquid is evaporated 

 in vacuo at the ordinary laboratory temperature. 



If one has a good vacuum-pump at one's disposal the liquid soon 

 begins to boil ; after some minutes the acetone is evaporated. A watery 

 fluid remains in which, besides other serum-substances, all the bili- 

 rubin is dissolved. Then the fluid is shaken 2 times or more with 

 aether to remove the fatty bodies as much as possible- 



These pass into the aether which is pipetted. The last traces of 

 aether are removed in vacuo. The aether may of course also be 

 removed by means of a separatory. Then a certain amount of chloro- 

 form e.g. 2 cm', are added, the fluid is slightly acidified with HCl 

 and shaken. The bilirubin then passes into the chloroform. By centri- 

 fugalization the watery fluid can easily be separated from the chloro- 

 form. The chloroform is washed thoroughly with water to get rid of 

 all the hydrochloric acid and centrifugalized once more, the water being 

 removed by means of a separatory or by pipetting. Traces of water, 

 however, remain mixed with the chloroform, which sometimes renders 



1) Hammarsten. Maly's Jahresber. 1878 II. 119. 



