Cxxxvi. the Isolation from Bile of a Pigment Having a Direct

THE pigments present in the blood-serum in jaundice were first differentiated by van den Bergh, who found that, whereas all icteric sera coupled with diazobenzene-p-sulphonic acid (Ehrlich's diazo-reagent) in the presence of alcohol, only certain of these sera would couple in simple aqueous solution. These diazo-reactions van den Bergh termed "indirect" and "direct" respectively, and it is now established beyond doubt that they are to be ascribed to two forms of bile pigment, the so-called indirectand direct-reacting pigments. Subsequent work has shown that these pigments differ so much in other respects as to justify the conclusion that they are of distinctly different types; the evidence on this point has been summarised by Hunter [1930]. Careful investigation of the conditions under which bilirubin couples with diazo-reagent and comparison of solutions of this pigment in serum with indirect-reaction sera led us to the conclusion that bilirubin is responsible for the indirect reaction [Griffiths and Kaye, 1930, 1, 2], a view first put forward by Collinson and Fowweather [1926] and later by Roberts [1928] and Hunter [1930]. In regard to the nature of direct-reacting pigment, however, there is still much difference of opinion. The views of earlier workers were presented in our previous papers, so that we shall confine our dttention to subsequent publications. Hunter [1930] found that if an alkaline solution of bilirubin is first buffered with disodium phosphate, it will couple with diazo-reagent in a distinctly acid medium. On the basis of this observation he suggests that direct-reacting pigment is sodium hydrogen bilirubinate, a view in accordance with that of Collinson and Fowweather [1926], who regarded the pigment as a salt of bilirubin, probably the ammonium salt. More recently, Fowweather [1932] has prepared the monoand di-sodium salts of bilirubin, and these, when dissolved in serum, were found to give only delayed diazo-reactions. This finding seems definitely to exclude the hypothesis of Hunter [1930], and of Newman [1928], that the pigment is a sodium salt. In this connection it is of interest to recall that van den Bergh and Muller [1916] observed that a neutral solution of bilirubin would not couple with diazo-reagent. Fowweather was unable to demonstrate conclusively that ammonium bilirubinate gives a direct reaction.