Studies on Bile Pigment Precursors in Erythrocytes Part 1. Micro-quantitative Analysis of Bile Pigment Precursors in Erythrocytes and its Alteration in Various Experimental Conditions

The method of micro-quantitative analysis of bile pigment precursors in erythrocytes was studied and various experimental observations were made for the elucidation of its physiological significance. The following results were obtained: 1. Studies of the fluorescent micro-quantitative analysis made it clear that bile pigment precursors in erythrocytes can be determined in the 0.1-5.0μg/ml biliverdin chloroform solution. Observing its absorption spectrum and fluorescent spectral characteristics, it was also made clear that all the bile pigment precursors were determined by the fluorescence of choleterin zincic complex salt. 2. Gardikas' method of extracting intracorpuscular bile pigment precursors under an anaerobic condition without adding ascorbic acid proved to be better than that of R. Lemberg of extracting by adding ascorbic acid under an aerobic condition. 3. Among normal erythrocytes of various animals were found no significant specific differences, the amount of bile pigment precursors being about 15μg/Hb. 4. When dog's erythrocytes were iucubated at 37℃. after the addition of ascorbic acid, bile pigment precursors in them attained to a remarkably great amount within several hours. This is considered to have resulted from the action of ascorbic acid in choleglobin formation in erythrocytes. 5. 4 hours after phenylhydrazine HCl was administered to a rabbit, the amount of bile pigment precursors in erythrocytes in circulating blood began to increase and attained to its maximum in 4-48 hours. The amount of easily split off blood iron also increased in parallel with it. 6. When canine blood was taken in A.C.D. solution and kept at 4℃., the amount of bile pigment precursors in erythrocytes gradually increased with the course of time, showing a sudden increase after 4 weeks. The amount of easily split off blood iron showed a similar variations. 7. In the erythrocytes of a phlebotomized anemic rabbit, the amount of bile pigment precursors decreased at the same time as reticulocytes showed a remarkable increase. The amount of easily split off blood iron showed a similar tendency of decrease. 8. From the above results the following conclusions were drawn. Bile pigment precursors which are usually observed in normal erythrocytes are increased by chemicals such as ascorbic acid and phenylhydrazine HCl, or by preservation, while in the case of anemia by phlebotomies where immature erythrocytes increase, the amount of bile pigment precursors decreases. Consequently, bile pigment precursors in erythrocytes physiologically increase with deterioration.