EASILY DETACHABLE IRON IN BLOOD 483 



iron set free is variable and depends on tlie strength of tlie acid used, stronger 

 hydrochloric acid liberating less than weak acid; weaker acid probably leaves 

 the "acid hematin" a stronger intramolecular peroxidase than does strong 

 acid.* Barkan {16o) has now accepted this explanation. As a result of this 

 many of the physiological deductions drawn from an estimation of easily 

 detachable iron now need revision. Some of the reagents which have been 

 used for the estimation of iron, particularly thioglycolic acid (2814), liberate 

 iron from hemoglobin i2o4T). 



Nevertheless. Barkan's assumption of the occurrence of a hemoglobin 

 derivative with easily detachable iron and closely related to bile pigments, is 

 probably correct, although this forms a much smaller percentage of the 

 erythrocyte iron than had been assumed. 1 to 2% of the erythrocyte iron 

 is set free in the presence of carbon monoxide. Small amounts of biliverdin 

 (a few micrograms per milliliter of erythrocytes) were obtained by Lemberg 

 and co-workers (1704J712) from hemolyzed erythrocytes, even if ascorbic 

 acid was added to the acid (6(5% acetic acid) used for the liberation of the 

 ironf; it has been shown (cf. Chapter VIII. Section G.) that ascorbic acid 

 prevents the intramolecular oxidation in the acidified oxyhemoglobin-mole- 

 cule. The biliverdin obtained from the erythrocytes can hardly have been 

 present as such in the cells, since the erythrocyte contains systems able to 

 reduce biliverdin to bilirubin (cf. Chapter XI, Section 8.). Barkan and 

 Walker (IGG) have observed an increase in plasma iron and bilirubin when 

 blood containing an anticoagulant was incubated at 37° C. Iron and bilirubin 

 are probably derived from a precursor present in the erythrocytes. Although 

 under pathological conditions, e.g., in animals receiving phenylhydrazine, 

 choleglobin can be demonstrated in the cells (cf. Chapter XI), Lemberg and 

 collaborators (1707,1710) observed in the normal erythrocyte only a weak 

 absorption band at 660 m/x after reduction with dithionite. 



It is clear that conclusions drawn from the estimation of easily detachable 

 iron can be interpreted only with the utmost caution. Large increases in 

 easily detachable iron can be considered as significant, but small variations 

 cannot. In experiments with radioactive iron, for example. Miller and Hahn 

 (1950) found that young, newly formed erythrocytes contained the same 

 amounts of easily detachable iron as the average of red cells of varying age. 

 They concluded from this that "easily detachable iron" is an artifact, and 

 that it cannot be formed by a gradual degradation progressing in the circu- 

 lating erythrocyte. While the former is true for the major portion of the 

 easily detachable iron, the latter has not been proved. In view of the large 

 variations in total easily detachable iron and the fact that the really interest- 



* According to Liebecq, Delbrouck, and Prijot (1738c), more iron is liberated with 

 small amounts of acid, when hem/globin is formed, than with large ones, when the 

 product is acid hematin. 



t Gardikas, Kench, and Wilkinson (979a) have recently claimed that no bile pigment 

 can be obtained from erythrocytes after acid treatment in the absence of ascorbic acid 

 if oxygen is excluded. It can be shown, however, that acid treatment of corpuscles 

 after saturation with carbon monoxide or anaerobically, in tJie absence of ascorbic 

 acid, gives the same yield of bile pigment (7-9 /u.g. per ml. of human or sheep cells). 

 In this instance the major part of the bile pigment is not biliverdin but a weakly basic 

 bilipurpurin found in the extracts with 20% hydrochloric acid (Lemberg, 16S7a). 



