These investigations having shown that inorganic iron is at least to some extent absorbed and carried to organs which take part in the production of hemoglobin, it became of especial importance to determine by long-continued feeding experiments whether the inorganic iron thus absorbed can take the place of food iron in the production of hemoglobin under normal conditions.

This question was studied by Hausermann in an extended series of experiments in Bunge's laboratory. The general plan of these experiments was to feed young animals from the end of the normal suckling period upon food poor in iron, usually milk and rice. One half of the animals, however, received ferric chloride in addition to this food. After the animals had been thus fed for from one to three months and had usually doubled in weight, they were killed, and the amount of hemoglobin in the entire body was estimated; also, in the case of small animals, the total amount of iron. Experiments were carried out in this way upon 24 rats, 17 rabbits, and 14 dogs. The results are summarized essentially as follows by Bunge: *

The rats all became highly anemic, for at the end of the experiment the percentage of hemoglobin was diminished to about half that of animals from the same litter which had received their normal food, namely, meat, flies, yolk of egg, fruit, and vegetables. The rats which had taken ferric chloride in addition to the milk and rice contained no more hemoglobin than those which had received milk and rice only. Moreover, the amount of iron was in each case the same. In one experiment alone, in which the addition of ferric chloride was continued for three months, was the iron found to be double as much in the animals which had received it as in those which had only milk and rice. But here again the proportion of hemoglobin remained the same in both instances. We thus see that some iron is absorbed if small doses of iron are persisted in for a long time, as well as if large amounts be suddenly administered. But this inorganic iron, when absorbed, is not utilized in the formation of hemoglobin to any appreciable extent, but remains unused in the tissues. Whether inorganic iron was absorbed in the experiments which lasted only from one to two months cannot be decided; it is possible that some of it was absorbed and was again eliminated in the same degree. Certainly no storing up nor increase of iron could be detected in the whole organism.

* Physiological and Pathological Chemistry, Blakiston's edition, Philadelphia, 1902, page 379.

The experiments on rabbits gave less decisive results. The average proportion of hemoglobin in the animals that received inorganic iron was somewhat higher than that in the animals which were fed on milk and rice only. But when the great individual differences between various animals are taken into consideration, too much importance must not be ascribed to this slight divergence. At any rate, the amount of hemoglobin in the control animal, which received its normal diet - fresh green cabbage, bran, etc. - was nearly twice as high as in the animal which received the inorganic iron.

The experiments upon dogs were not attended with decisive results, as dogs are not suitable animals for these experiments, owing to the variation in individuals. Moreover, the growth of these animals after the period of lactation is at a much slower rate, and their appetite is so enormous that they might readily be able to assimilate sufficient iron for hemoglobin formation even from a material so poor in iron as milk. In fact, Hauser-mann found the largest proportion of hemoglobin in a dog which had been fed exclusively upon milk. The animals which received ferric chloride in addition to a milk diet certainly contained no more hemoglobin than animals from the same litter which were fed on meat and bones.

Abderhalden, following Hausermann, studied the subject even more exhaustively. In order to ascertain whether and to what extent sulphides normally exist in the alimentary canal, - a question of special importance in connection with one view of the mode of action of inorganic iron, - Abderhalden killed and examined rats, mice, cats, dogs, guinea pigs, and rabbits in the following way: Immediately upon killing the animal, the abdomen was opened and the intestinal tract from the esophagus to the rectum was ligated in sections. The contents of each section were then removed and tested qualitatively for sulphides. Hydrogen sulphide was obtained from the contents of the large intestine, but not from those of. the small intestine nor of the stomach. Hence, if inorganic iron acts by improving the absorption of food iron, it must do so in some other way than by simply preventing its precipitation as sulphide, since this would not occur in the small intestine, where the principal absorption of iron takes place. The next step in the investigation was to study by microchemical methods the absorption of inorganic iron, its behavior in the body, and its elimination. Experiments were made upon 49 rats from 7 litters, 14 guinea pigs from 6 litters, 12 rabbits from 2 litters, 10 dogs from 3 litters, and 6 cats from 2 litters.

From all of these experiments, Abderhalden concluded that the complicated iron compounds of the normal food, the iron in the form of hemoglobin, and hematin, and the inorganic iron, were all absorbed in the same general way, stored in the same organs, and eliminated by the same paths.

In studying the utilization by the body of the different forms of iron, Abderhalden fed animals from the end of the suckling period, or, in the case of guinea pig, from birth, on food poor in iron, and divided each litter into two groups, one of which was given inorganic iron in addition. After a sufficient time the animals were killed, and the total hemoglobin in the body of each was estimated. Experiments of this kind were made upon 48 rats, 44 rabbits, 14 guinea pigs, 17 cats, and 11 dogs. The animals fed with food poor in iron plus an addition of inorganic iron were unable to produce as much hemoglobin as those receiving normal food.

In these experiments, Abderhalden had noticed some facts which indicated that the favorable influence of inorganic iron upon metabolism and blood formation was greater on a diet rich in food iron than when the amount of food iron was kept small. In order to test this, experiments were made with 66 rats, 10 rabbits, and 14 guinea pigs, in the manner already described, but with diets arranged to bring out this particular point. These experiments led to the conclusion that the greater the quantity of food iron present, the greater the influence of the inorganic iron upon the hemoglobin formation.

Abderhalden's experiments also showed that the production of hemoglobin was not stimulated indefinitely by inorganic iron, but only for a short time, and he concluded that, while inorganic iron may be absorbed and may favorably influence blood formation, it is not used as material for the production of hemoglobin. It has also been found clinically that medicinal iron gives better results when used intermittently than when used continuously, which indicates that the action is due to stimulation rather than to the inorganic iron actually going to form hemoglobin.

The results obtained by Tartakowsky * were more favorable to the view that hemoglobin may be formed from inorganic iron. He found that young growing animals fed on rice and milk gradually became anemic and finally ceased to grow; but that when inorganic iron was added to the rice-milk diet the blood regained its normal iron content and the animal soon began to grow again. From such experiments together with a large number of microchemical observations, Tartakowsky concludes that medicinal (inorganic) iron is assimilated like food iron and serves in the same way for the production of hemoglobin and the other organic iron compounds of the body. He further insists that Abderhalden's experiments should also be interpreted in the same way, since in many cases the animals which received inorganic iron in addition to their food formed more hemoglobin than the control animals.

More recently, Schmidt † has described some interesting experiments upon mice with a similar iron-poor rice-and-milk diet. According to Schmidt this diet did not cause anemia in adult mice; but the offspring of mice which had been kept on such diet seemed to lack the normal reserve store of iron, and by continuing the milk-rice diet to the third generation there were obtained what this investigator describes as "iron-free families" of mice. In these the red blood cells were very poor in hemoglobin. From such a family of mice two sisters seven months old were selected; one was continued on the milk-rice diet alone while the other was fed medicinal iron (Ferrum oxyda-tum saccharatum) in addition for eleven days; then both were killed and examined. The first showed the typical anemic condition of these "iron-free families," the hemoglobin number and number of red blood cells being both less than half of the normal; while in the second mouse, which had received medicinal iron for eleven days, the hemoglobin number and number of red blood cells were both about twice as high as in the first. This is held by Schmidt to show that medicinal iron does not merely stimulate the blood-forming organs to greater activity but does itself enter into hemoglobin formation.

* Archiv fur die gesamte Physiologie, Vol. 100, page 586; Vol. 101, page 423 (1903, 1904).

† Verhandlungen der Deutsches Pathologisches Gesellschaft, Vol. 15, page 91 (1912).

It is difficult to determine how much weight should be given to the findings of Tartakowsky and of Schmidt as opposed to the more extended and more quantitative experiments of Hauser-mann and of Abderhalden.

While it cannot yet be stated positively that inorganic iron is or is not used by the animal body as material for the production of hemoglobin, the best medical opinion appears to support the conclusion reached by Abderhalden, that hemoglobin is derived essentially from the organic iron compounds of the food, while inorganic iron acts mainly if not entirely as a stimulus. This view is strongly supported by Von Noorden in his treatise on chlorosis in Nothnagel's Encyclopedia of Practical Medicine, and Ehrlich and Lazarus, writing on anemia in the same work, state:

"It is not very probable that the (medicinal) iron stored by the liver and spleen is directly employed in the formation of hemoglobin; on the contrary, the assumption first suggested by Von Noorden seems much more plausible, namely, that the iron exercises a direct irritative action on the function of the blood-making organs."