BILIRUBIN IN BLOOD PLASMA 547 



tized sulfanilic acid in a manner different from that of bilirubin in 

 patients with obstructive jaundice. In obstructive jaundice the red 

 azo dye was formed with the reagent alone — the bilirubin reacted 

 "directly" — while in the first-mentioned cases it was formed only 

 after the addition of alcohol ("indirect reaction"). On the basis of 

 these experiments and of those of McNee and Lepehne {1719), 

 Aschoff (85-88) suggested that "indirect bilirubin" is produced by 

 the breakdown oi hemoglobin in reticuloendothelial cells, and is 

 changed to "direct bilirubin" by the excreting liver cell; in cases of 

 obstruction of the bile duct the latter is dammed back into the blood 

 stream. 



This hypothesis has led to a veritable flood of investigations and hypotheses 

 to explain the difference between "direct" and "indirect" bilirubin. There 

 are few fields in biochemistry in which so much work has been done with so 

 little success. The subject has been reviewed repeatedly (178,1913,3031; 

 2989, p. 2474). We still have no exact knowledge of the mechanism of the 

 coupling reaction (cf. Chapter IV, Section 4.3.), nor of the basis of the differ- 

 ence between the two types of reaction. Watson's assumption that the 

 coupling occurs in a furan ring of bilirubin (2989, p. 2476) was based on an 

 erroneous formula of Fischer, long abandoned by Fischer himself. 



"Indirect bilirubin" is made to react with the diazo reagent not only by 

 alcohol, but also by caffeine (11^14, 1415, 1719; cf. also Chapter IV, Section 

 9.1.), while directly reacting sera or urine become indirectly reacting on 

 standing or boiling. 



The two types of bilirubin also differ in other properties, although the 

 coordination of these differences with the manner of reaction with the diazo 

 reagent is not always straightforward, and there are contradictory claims. 

 "Indirect bilirubin" is more readily extracted from the serum by chloroform 

 (56,53^,1066,1203,2590), while ether and butanol extract "direct bilirubin" 

 preferentially (4-87,2858). The "indirect bilirubin" of hemolytic anemia is 

 less readily excreted into the urine in large amounts than the "direct bili- 

 rubin" of obstructive jaundice (1843,2199,2339,3037 ;2989, p. 2559). 



These differences have been variously explained by assuming two chemi- 

 cally different bilirubins, two tautomeric forms of bilirubin, different amounts 

 of bilirubin, a different kind of solution of bilirubin, influences of pH and of 

 substances present in the plasma such as lipoids, cholesterol, or bile acids. 

 All these hypotheses have been contradicted in turn. 



It is certain that the bilirubin in both forms is one and the same, and is 

 identical with the bilirubin from bile, and there is also no evidence for the 

 existence of tautomerism (cf. Chapter IV, Section 2.). The absorption 

 spectrum of serum bilirubin differs from that of a solution of bilirubin in 

 phosphate buffer of the same pH, the maximum of the absorption of the latter 

 being at 425 m/Lt, while the maximum in human serum is 460 myu, in horse 

 serum, 470 m/x (1213, p. 153; 1223). The absorption spectra of "direct" 

 and "indirect" bilirubin in serum are very similar; slight differences have 



