548 XI. HEMOGLOBIN CATABOLISM, I 



been noted by Heilmeyer and Krebs {1216) and by Davis and Sheard {o37), 

 but these are given as being in the opposite direction in the two papers. 



It is not even certain how bihrubin itself reacts under the conditions 

 present in the serum. Davies and Dodds {oSJi) found a solution of bilirubin 

 in phosphate buffer added to serum to react directly, while Barron {178) 

 found sodium bilirubinate to react directly, but indirectly if added to serum. 

 The former has also been found by Fiessinger and co-workers (see below). 



'The confusion has been somewhat cleared by the proof that both 

 'direct" and "indirect" bilirubin of the blood are protein compounds 

 of bilirubin {213,215487,920,2132). This proof is based on ultra- 

 filtration, ultracentrifugation, and cataphoresis experiments. Com- 

 bination of bilirubin with globin had, without evidence, already been 

 assumed by several authors {233,639,1331,1742,1223,2623); it has 

 now been supported by experimental evidence by Fiessinger and 

 co-workers {748-750,2162). They observed that a solution of sodium 

 bilirubinate when added to globin and neutralized with acid, reacted 

 "indirectly" and that the bilirubin could be extracted with chloro- 

 form. If serum albumin was used instead of globin, a direct reaction 

 was obtained, and the bilirubin could not be extracted with chloro- 

 form. Direct bilirubin (from the serum of a patient with cancer of 

 the pancreas) could be made chloroform-extractable by being incu- 

 bated with globin. The protein, obtained after chloroform extraction 

 from sera giving an indirect reaction, could be shown to be globin 

 by coupling it with hematin to hemoglobin. If these observations can 

 be confirmed, "indirect bilirubin" is bilirubin still combined with 

 globin, while "direct bilirubin" is bilirubin which, after having been 

 set free in the liver, has recombined with serum albumin, in the same 

 way as hematin set free from hemoglobin recombined with serum 

 albumin to form methemalbumin. 



Pedersen and Waldenstrom have provided evidence against this 

 view {2132). They found no difference between the variation of 

 electrophoretic mobilities of "direct" and "indirect" bilirubin with 

 pH, both behaving in the same way as serum albumin and not as 

 globin. Recently, Ilobscheit-Robbins and co-workers {2292), study- 

 ing the electrophoretic mobility pattern of the plasma proteins, came 

 to the conclusion that a modified globin is present in the plasma after 

 intraperitoneal injection of hemoglobin {cf. Section 10.4.). It may 

 well be this protein which, combined with bilirubin, constitutes the 

 "indirect" bilirubin of normaK and hemolytic sera.* 



* While Watson {2991a) has come to the same conclusion as the French workers, our 

 own experiments have failed to confirm important points of their evidence. Recently, 



