Indirect bilirubin. I. Differential character of indirect bilirubin and direct acid bilirubin. II. Spontaneous variations of direct and indirect bilirubin in various conditions of preservation. HI. The action of oxidizing agents on the behavior of bilirubin to direct diazo reaction. IV. The nature of indirect bilirubin
Gastaldi, F.; Delrio, G.; Testoni, F.
Bollettino della Societa Italiana di Biologia Sperimentale 23(7): 689-698
I. Fresh bile from dog gall bladder contains a small fraction of bilirubin which can be extracted by chloroform and which reacts indirectly with the diazo reagent. With the addition of NaOH to a bile fraction containing bilirubin which acts both directly and indirectly with the reagent, the chloroform extractable fraction disappears. However, neutralization to pH 7.5 with acetic acid again permits the chloroform, but not ether, extraction of the indirectly reacting bilirubin, and with neutralization to pH 6.4 a fraction can be extracted with ether and with chloroform which reacts positively with the reagent. Therefore, the view that direct bilirubin is in the acid state and indirect bilirubin in the salt state, is untenable.[long dash]II. Bilirubin was detd. in the unhemo-lyzed blood of 11 icteric patients immediately and after 3, 6, and 24 hrs., 2, 5, and 10 days. Similarly bilirubin was detd. in dog bile immediately and 1, 2 and 3 days later, both with the sample in contact and out of contact with air. In the blood samples and in the bile in contact with air, the proportion of indirectly reacting bilirubin increased as the total decreased. The direct bilirubin decreased and the indirect bilirubin remained practically constant in the bile out of air contact. The authors consider the possibility of the 2 reactions of direct bilirubin to biliverdin and to indirect bilirubin being oxidative processes.[long dash]III. Bile freed of indirect bilirubin by chloroform extraction was treated with dilute H2O2 with complete conversion to biliverdin and indirect bilirubin extractable with chloroform. Analogous results were obtained with icteric serum with a third of the direct bilirubin being converted to indirect bilirubin. Nitric acid oxidized bile to biliverdin and to a dipyrrol compound. Indirect bilirubin could be obtained as an intermediate compound. With ferric chloride biliverdin was formed to the exclusion of indirect bilirubin. [long dash]IV. Data on the chemical nature of indirect bilirubin are reviewed and discussed.