Studies on Intermediate Products of Bile Pigment Formation Utilizing Rapioactive Iron in Vitro Part 1. Chromatographic Studies on Intermediate Products from Pyridine-hematin to Bile Piment

Traceable Fe 55 hemin was dissolved in 20% pyridine water solution, then 1-ascorbic acid was reacted in absence of oxygen. Column chromatography was observed on the reaction mixture of each reacting time. Studies were made on the water solution of the divisible absorption layers and following deductions were obtained. 1) Hourly observations of chromatogram were always in 4 layers and their respective colors and distributions were different. Additionally the fouth layer, due to the failure of retaining it in the absorption column in spite of various manipulations attempted, was collected as a gravity flow effluent. 2) Distribution of the total iron was different with respective layer and reacting time, but there was iron which would not dissolve in water due its remaining in the absorption column unber ionic combination and with increased with the reaction progress. 3) Total iron in the first layer was very small i. e. traceable degree and the greater part of it was easily split off iron. Biliverdin was undetectable therefore absorption curve worth mentioning was obtainable. 4) Conversely with the hourly decrease of total iron in the second layer, easily split off iron increased with the reaction progress. The amount of Biliverdin was highest at the end of four hours. In the absorption curve the peak absorptions of 610 mμ was observed right after the initiation of reaction and after one hour which was believed to be due to pyridine-hemichromogen being absorbed. 5) Total iron of the third layer was exceedingly small and most part of it was easily split off iron. The quantity of Biliverdin was highest at the end of fourth hour. Absorption curve was not obtainable. 6) For the fourth layer total iron showed the peak at the end of four hours but the value for the easily split off iron was relatively high throughout and little deflection was observed for all reaction progress. Biliverdin content was highest in this layer and highest at the fourth hour. With reference to the absorption curve at the initial reaction stage 3 maximum peaks of 656, 530 and 500 mμ were observed. This was parallel with protoverdohemochrome. With the progress of reaction this was equal with the 656 mμ of pyridineverdohemochromogen. 7) The quantity of easily split off iron, that of ron just after the addition of hydrochloric acid and so called free iron was parallel but value of easily split of iron content was higher throughout. Moreover the quantity of easly split off iron and Biliverdin quantity did not show parallel relation to each other. 8) Permeating liquid could be observed after 2 hours reaction. The colour of the liquid was light strow at the begining and as the time elapsed brownish tinge increased. Also the quantity of total iron increased with time lapse but notable absorption curve was not obtainable. Presence of Biliverdin could not be proven qualitatively