Studies on Intermediate Products of Bile Pigment Formation Utilizing Rapioactive Iron in Vitro Part 2. Chromatographic Studies on Intermediate Products from Hemoglobin to Bile Pigment

The following deduction were obtained, reacting traceable Fe(59) hemoglobin solution with 1-ascorbic acid under absence of oxygen, and subsequently observated the formed intermediate products in the column chromatography as the time elapsed. 1) The column stratified into four layers in 4 hours after initial reaction and into 3 layers 8 hours after start of reaction. Colors and distribution of strata differed at corresponding reacton times. 2) Total iron distribution differred with corresponding absorption layer and reaction time. 3) The hourly increase of total iron in the first stratum was due mostly to increase of easily split off iron. 4) Total iron content of second layer was small and hourly change was minute, however at the start of the reaction the presence of easily split off iron was exceedingly large. Although easly split off iron content reduction was fast in comparison with other layers it appeared as though this layer was the initiator of reaction progress. 5) Total iron content was largest in the third layer, but the quantity of easily split off iron was exceedingly small and hourly change was lacking. 6) Against the fast rate of reduction of total iron content of the fourth layer, the easily split off iron increased at a fast rate. 7) Easily split off iron content was always higher than the iron content immediately after the hydrochloric acid addition or so called free iron. 8) The presence of the so called free iron is obvious, but it was not clear whether it was the natural free iron it was due to trichloro-acetic acid. 9) Differing from other layers, the iron in the first layer was harder to ionize. 10) Biliverdin was analysed from all layers but parallel between its content and that of easily split off iron was not recognized.