Antianaemic composition and a process for producing the same

A process for producing an organoiron(II) compound-containing antianaemic composition which comprises cultivating a yeast in a saccharide-containing nutrient medium therefor in the presence of an iron compound to form a cultured broth comprising an organoiron(II) compound, alcohol and water and removing the alcohol from the cultured broth to an extent that the resulting cultured broth has an alcohol content of less than about 1% by volume, and an antianaemic composition produced thereby. The antianaemic composition of the present invention is very stable, and excellent in absorbability into a living body and incorporation of iron into hemoglobin.

This invention relates to an antianaemic composition and a process for 
producing the same. More particularly, the present invention is concerned 
with an organoiron(II) compound-containing antianaemic composition which 
can be well absorbed into the living body and is chemically stable and the 
production of the same by a process comprising cultivating a yeast in a 
saccharide-containing nutrient medium in the presence of an iron compound, 
followed by removal of alcohol. 
Since iron is a major constituent of hemoglobin, various kinds of iron 
compounds are used as an antianaemics. Heretofore, a considerable number 
of researches have been made on the absorption of iron into a living body 
through the stomach and intestine organs. As a result, it is now known 
that Fe.sup.++ can be well absorbed in the human organs but Fe.sup.+++ 
is not. Accordingly, divalent iron compounds such as ferrous sulfate are 
most frequently used as a chalybeate. When using divalent iron compounds 
as a chalybeate, however, special pharmaceutical techniques such as 
coating should be applied because they are readily oxidized to trivalent 
iron compounds. It is also known that an iron complex with an brganic 
acid, chelating agent, sugar, amino acid or the like is better absorbed 
into a living body than inorganic iron compounds. Practically, an organic 
acid salt of a divalent iron such as ferrous fumarate or ferrous 
succinate, or a divalent iron chelate has been used for the remedy of a 
patient suffering from anaemia. Such ferrous compounds, however, still 
have a disadvantage that they tend to easily be oxidized to the 
corresponding ferric compounds during the storage or in the living body 
even after administration thereof to a patient. 
On the other hand, wine has long been believed to be effective for the 
remedy of a patient suffering from anaemia, but any elucidation has not 
yet been made as to whether the iron values contained in wine are really 
effective as a chalybeate, or as to in what state the iron values in wine 
exert an antianaemic effect even if they are admitted to be effective. 
Further, there is not any accepted conviction as to where the iron values 
contained in wine originate. Furthermore, the iron content in the wine put 
on the market widely varies. Therefore, drinking wine does not always 
ensure absorption of iron into the living body. 
The present inventor has made extensive and intensive researches on the 
iron values contained in wine. Illustratively stated, the present inventor 
has conducted the analysis of an iron content of the raw material (grape 
juice) for wine and found that an amount of iron to which the iron content 
present in the wine put on the market can be attributed is not contained 
in the ordinary raw material for wine and that most of the iron values 
present in the wine put on the market is due to contamination during the 
fermentation stage. Further, the present inventor has studied the state of 
iron values present in wine, which is effective for the use as a 
chalybeate. Based on the findings,study and the subsequent studies for 
developing an antianaemic agent, the present invention has been made. 
Accordingly, it is an object of the present invention to provide a process 
for producing an organoiron(II) compound-containing antianaemic 
composition which can be efficiently absorbed into a living body and 
chemically stable. 
It is another object of the present invention to provide a process of the 
above-mentioned kind which can be carried out easily at low cost. 
It is a further object of the present invention to provide an 
organoiron(II) compound-containing antianaemic composition produced by a 
process as mentioned above.

In one aspect of the present invention, there is provided a process for 
producing an organoiron(II) compound-containing antianaemic composition 
which comprises: (1) cultivating a yeast in a saccharide-containing 
nutrient medium in the presence of an iron compound to form a cultured 
broth comprising an organoiron(II) compound, alcohol and water, and (2) 
removing the alcohol from the cultured broth to an extent that the 
resulting cultured broth has an alcohol content of less than about 1% by 
volume. The kind of a yeast is not critical, but a wide variety of general 
ordinarily employed in fermentation may be employed. Preferable genus is 
Saccharomyces or Metschnikowia. Examples of species of yeast which may 
preferably be employed are shown in Table 1. 
TABLE 1 
______________________________________ 
Species Strain 
______________________________________ 
1. Saccharomyces cerevisiae 
OC-2 
2. Metschnikowia pulcherrima 
K-427 
3. Saccharomyces chevalieri 
IAM-4861 
4. Saccharomyces bayanus 
PW-15 
5. Saccharomyces italicus 
PW-27 
6. Saccharomyces fermentati 
WF-107 
7. Saccharomyces rosei 28-t 
8. Saccharomyces heterogenicus 
PW-25 
______________________________________ 
Note: The species and strains listed in Table 1 are all known. Of the 
abovementioned species, Saccharomyces cerevisiae is most preferred becaus 
it can produce a large quantity of a fraction B having an excellent 
absorbability through the intestine as will be explained later. 
Examples of the saccharide-containing nutrient medium to be employed in the 
present invention include fruit juice, semi-synthetic culture medium and 
synthetic culture medium. Specific examples of the fruit juice include 
grape juice (or must), apple juice and the like. Of substances of 
saccharide, saccharose such as sucrose, glucose, fructose or the like may 
more preferably be employed. Preferred examples of semi-synthetic medium 
and synthetic nutrient medium are given in Tables 2 and 3, respectively. 
TABLE 2 
______________________________________ 
(semi-synthetic) 
______________________________________ 
D-glucose 100.0 g 
(NH.sub.4).sub.2 SO.sub.4 
5.0 g 
KH.sub.2 PO.sub.4 1.0 g 
MgSO.sub.4.7H.sub.2 O 0.50 g 
NaCl 0.10 g 
CaCl.sub.2.2H.sub.2 O 0.10 g 
Yeast extract 1.0 g 
Distilled water 1000 ml 
Initial pH 6.0 
______________________________________ 
TABLE 3 
______________________________________ 
(synthetic) 
______________________________________ 
D-glucose 150 g 
(NH.sub.4).sub.2 SO.sub.4 
3.5 g 
KH.sub.2 PO.sub.4 1.0 g 
MgSO.sub.4.7H.sub.2 O 0.50 g 
NaCl 0.10 g 
CaCl.sub.2.2H.sub.2 O 0.1 g 
H.sub.3 PO.sub.3 500 .mu.g 
CuSO.sub.4.5H.sub.2 O 40 .mu.g 
KI 100 .mu.g 
FeCl.sub.3.6H.sub.2 O 242 mg 
MnSO.sub.4.H.sub.2 O 400 .mu.g 
Na.sub.2 MoO.sub.4.2H.sub.2 O 
200 .mu.g 
Biotin 2 .mu.g 
ZnSO.sub.4.7H.sub.2 O 400 .mu.g 
Calcium pantothenate 400 .mu.g 
Inositol 2000 .mu.g 
Thiamine.HCl 400 .mu.g 
Distilled water 1000 ml 
Initial pH 7.0 
______________________________________ 
The kind of artificial nutrient medium, of course, is not limited to the 
above-mentioned examples, and any formulations employable in an ordinary 
fermentation using saccharide-containing nutrient medium may be employed. 
As the iron compound, any of those which can be ionized with respect to 
iron may be employed. As specific examples, there can be mentioned 
inorganic acid salts of iron such as ferric chloride, ferrous sulfate and 
ferric sulfate and organic acid salts of iron such as iron citrate and 
iron tartrate. Iron powder is also useful, but disadvantageously needs a 
long period of time for its dissolution into a culture medium. From the 
viewpoints of solubility, availability and non-toxicity, the 
above-mentioned iron compounds are preferred. Such an iron compound, when 
it is of trivalent iron, is converted to produce ferrous ions during the 
fermentation. The amount of the iron compound to be added is not critical, 
but even if the amount of iron added is increased, the amount of iron in 
the product is not necessarily increased in proportional relationship. 
Further, the addition of too large an amount of the iron compound 
adversely suppresses the progress of fermentation. On the other hand, the 
use of too small an amount of the iron compound cannot exert the intended 
effect. Usually, the iron compound may be employed in the nutrient medium 
at a concentration of about 10 to about 70 ppm, more preferably about 30 
to about 50 ppm in terms of amount of iron. 
In practicing the present invention, whether the fruit juice, 
semi-synthetic culture medium or synthetic medium is used, the yeast may 
be employed at a yeast population of about 5.times.10.sup.5 to about 
3.times.10.sup.6 /ml of culture medium. 
Further fermentation conditions are given as follows. (1) In the case of a 
culture medium of fruit juice, the cultivation of a yeast can be carried 
out according to a customary process for the production of wine. For 
example, the fruit juice, e.g., grape juice or apple juice, as such, or 
after a saccharide such as glucose, fructose, sucrose or the like is added 
thereto in such an amount as will exhibit a refraction saccharide degree 
of about 20 to about 25, is subjected to treatment with a small amount of 
a germicide (e.g., about 100 ppm of potassium metabisulfite) to kill 
contaminating microorganisms such as wild yeast. Then, the cultivation may 
usually be conducted at room temperature to 30.degree. C. under 
atmospheric pressure for about 15 days. (2) In the case of a 
semi-synthetic culture medium or a synthetic culture medium, the 
cultivation may be conducted at about 18 to about 30.degree. C. under 
stationary condition or anaerobic condition. The amount of a saccharide in 
the nutrient medium is not critical. From a viewpoint of provision of 
better conditions of the desired fermentation, the saccharide may 
generally be incorporated into the nutrient medium in an amount of about 5 
to about 25% by weight, more preferably about 10 to about 15% by weight 
based on the nutrient medium. 
The pH of the artificial nutrient medium has initially a value of about 6 
to about 7. However, 3 to 4 hours after the initiation of fermentation, 
the pH value rapidly decreases to 3.6 to 3.8 (This value does not change 
thereafter) and then the rate of fermentation remarkably increases. 
Fermentation is continued for a period of from about 10 to about 20 days. 
The resulting cultured broth, which contains an organoiron(II) compound, 
alcohol and water, is subjected to removal of alcohol at a temperature not 
exceeding 30.degree. C. under a pressure of about 15 to 20 mmHg to such an 
extent that the alcohol concentration of the cultured broth is decreased 
to less than 1% by volume. The removal of alcohol from the cultured broth 
may be efficiently effected by any of the commonly-known techniques, for 
example, using a rotary evaporator. 
The alcohol concentration of the cultured broth may be traced by 
determining the specific gravity of each aliquot taken out from the 
cultured broth. Illustratively stated, an aliquot is taken out from the 
cultured broth, and the specific gravity of the aliquot at a predetermined 
temperature is measured. Then, the alcohol concentration corresponding to 
the specific gravity is read on the graph obtained by plotting the 
specific gravity of each solution having a known alcohol concentration 
against the alcohol concentration. 
The method for determining the alcohol concentration is not limited to the 
above-explained method, but in fact any other appropriate method, 
including chromatography, can be employed. 
As mentioned above, the cultured broth is subjected to removal of alcohol 
to such an extent that the alcohol concentration of the cultured broth is 
decreased to less than 1% by volume. The reason is that if the cultured 
broth having a high alcohol concentration of 1% by volume or more is 
continually administered as an antianaemic composition to a patient for a 
prolonged period of time, it adversely affects the health of the patient. 
The antianaemic composition produced by the present process can, first, be 
obtained in the form of a cultured broth. The cultured broth may then be 
separated into a precipitate and a supernatant, for example, by 
centrifugation or the like. The supernatant is also one form of the 
present antianaemic composition. In any of them, there is contained a 
water-soluble organoiron(II) compound which is believed to be an organic 
complex of divalent iron and is a fermentation product of iron formed by 
the initially added iron compound's undergoing a chemical reaction during 
the cultivation or fermentation. Both the cultured broth and the 
supernatent as such can be administered. For example, when the culture 
medium is grape juice, they can be enjoyed by people as an 
organoiron(II)-containing elixir. The concentrates may be ultimately 
concentrated to dryness, and the resultant as such or, if desired, 
together with a reducing additive such as Vitamin C, may be formulated 
into the adequate form for oral administration, for example, a tablet, 
capsule, powder, granule or fine granule form. If desired, these dosage 
forms may be easily prepared according to conventional technique and may 
comprise commonly employed excipients, binding agents, disintegrators, 
glidants and other pharmaceutical agents. As the excipient, binding agent 
and/or disintegrator, there may be, for example, mentioned 
microcrystalline cellulose, wheat starch, sugar, lactose, gum arabic, 
tragacanth gum, carboxymethylcellulose and so on. As the glidant, there 
may be given, e.g., magnesium stearate and talc. Tablets may be also 
coated according to conventional coating procedures and any commonly 
employable coating materials such as, for example, shellac, 
ethylcellulose, hydroxymethylcellulose, polyvinyl pyrrolidone, titanium 
dioxide and the like may be favorably applied for such purposes. The 
dosage may vary depending upon ages, severities and body weights of 
patients, but the present antianaemic composition may be usually 
administered in a daily dose of from about 5 mg to about 100 mg in terms 
of amount of iron for adults, if necessary, in divided dosage forms. 
Thus, in another aspect of the present invention, there is provided an 
organoiron(II) compound-containing antianaemic composition produced by a 
process comprising: 
(1) cultivating a yeast in a saccharide-containing nutrient medium in the 
presence of an iron compound to form a cultured broth comprising water, 
alcohol and an organoiron(II) compound, and 
(2) removing the alcohol from the cultured broth to an extent that the 
resulting cultured broth has an alcohol content of less than about 1% by 
volume. 
As stated before, from the chemical analysis, it is believed that the 
active ingredient of the present antianaemic composition is a divalent 
iron-containing organic complex composed only of elements Fe, C, H and O 
whose molecular weight is in the range of about 10.sup.2 to about 
5.times.10.sup.3. As is apparent from Experiment which will be given 
later, it is clearly recognized that the organoiron(II) in the present 
antianaemic composition is much more effective in capability of being 
absorbed in the living body of a human being and animals and incorporation 
into hemoglobin than the conventional chalybeates. Furthermore, it is 
clearly recognized that the organoiron(II) in the present antianaemic 
composition is remarkably stable as compared with divalent iron compounds 
such as ferrous chloride, ferrous sulfate, ferrous fumarate, ferrous 
succinate and ferrous tartrate. 
When the supernatant as mentioned before is subjected to chromatographic 
fractionation, for example, by means of Amberlite XAD-2(available from 
Rohm and Haas Co., U.S.A), it is separated into two fractions, namely, 
Fraction A and Fraction B. In this connection, Fraction B has a relatively 
low molecular weight but a high absorbability into the living body as 
compared with Fraction A. 
In still another aspect of the present invention, there is provided a 
method of increasing an iron content in blood which comprises 
administering to a patient an effective amount of an organoiron(II) 
compound-containing antianaemic composition produced by a process 
comprising: 
(1) cultivating a yeast in a saccharide-containing nutrient medium in the 
presence of an iron compound to form a cultured broth comprising an 
organoiron(II) compound, alcohol and water, and 
(2) removing the alcohol from the cultured broth to an extent that the 
resulting cultured broth has an alcohol content of less than about 1% by 
volume. 
The present invention will be illustrated in more detail with reference to 
the following Examples, which should not be construed to be limiting the 
scope of the present invention. 
EXAMPLE 1 
Sugar was added to grape juice from Koshu grape (produced in the vicinity 
of Kofu-city, Japan) in such an amount as would give a refraction 
saccharide degree of 24. To the mixture was added potassium metabisulfite 
in an amount of 100 ppm in terms of amount of sulfurous acid. Then, 
FeCl.sub.3 was added in an amount of 50 ppm in terms of amount of iron, 
and a yeast mash of Saccharomyces cerevisiae OC-2 was added in an amount 
of 3% by volume based on the grape juice. The mixture was subjected to 
fermentation at 20.degree. C. for 15 days. The resulting fermentation 
product was subjected to removal of alcohol at 25.degree. C. under a 
reduced pressure of 15 mmHg, using a rotary evaporator, to an extent that 
the alcohol concentration of the cultured broth was decreased to 0.5% by 
volume to obtain an antianaemic composition. The iron content of the 
composition was 31 ppm. 
EXPERIMENTS 
Animal tests were conducted to show the effectiveness of the iron values in 
the wine prepared according to the present invention. 
(1) Preparation of Test Sample 
(a) Wine according to the present invention 
Sugar was added to grape juice from Koshu grape in such an amount as would 
give a refraction saccharide degree of 24.To the mixture was added 
potassium metabisulfite in an amount of 100 ppm in terms of amount of 
sulfurous acid. On the other hand, .sup.59 FeCl.sub.3 was added to a 
solution of ferric chloride in 0.1N hydrochloric acid in such an amount as 
would give a specific radioactivity of 6.25 .mu.Ci/50 .mu.gFe, and the pH 
of the mixture was adjusted to pH 3by adding an aqueous 0.1N sodium 
hydroxide solution. This was added to the above-prepared grape juice 
medium in an amount of 50 ppm in terms of amount of iron. Then, a yeast 
mash of Saccharomyces cerevisiae OC-2 was added in an amount of 3% by 
volume based on the iron-containing grape juice medium. Fermentation was 
conducted at about 20.degree. C. After completion of the intended 
fermentation, the mixture was subjected to centrifugation at 10,000 rpm 
for 10 minutes to obtain wine containing .sup.59 Fe. Removal of alcohol 
was performed under reduced pressure at 30.degree. C. to an extent that 
the alcohol concentration of the wine was decreased to 0.5% by volume, and 
test samples respectively containing 10, 40, 80 and 250 .mu.gFe/ml were 
prepared by addition of distilled water. 
(b) Ferrous sulfate solution (Comparative) 
Put into the .sup.59 FeCl.sub.3 solution with a specific radioactivity of 
12.5 .mu.Ci/.mu.gFe was ascorbic acid in an amount of 0.05.mu. mol per 
.mu.g of iron to give divalent iron. By mixing this with an aqueous 
ferrous sulfate solution, comparative test samples having 5 .mu.Ci/10, 40, 
80 and 250 .mu.gFe/ml and a pH value of 3 were obtained. 
(c) Ferric chloride solution (Comparative) 
Put into the .sup.59 FeCl.sub.3 solution was an aqueous ferric chloride 
solution, and the pH of the mixture was adjusted to pH 3 by adding an 
aqueous sodium hydroxide solution. Comparative test samples having 5 
.mu.Ci/10, 40, 80 and 250 .mu.gFe/ml were obtained. 
(d) Concentrated wine-ferrous sulfate mixture (Comparative) 
Wine (in which the iron originating in grape juice is present in an amount 
of about 1 ppm) prepared in the same procedures as described in Example 1 
except that FeCl.sub.3 was not added was concentrated to 1/4 in volume. By 
mixing this with the ferrous sulfate solution (prepared according to the 
above-mentioned procedures and having 10 .mu.Ci/80 .mu.gFe/ml) at a ratio 
of 1:1 by volume, a comparative test sample having 5 .mu.Ci/40 .mu.gFe/ml 
was obtained. 
(2) Administration 
Five-week old male Sprague-Dawly rats (available from Nippon Kurea K.K., 
Japan) were subjected to preparatory breeding for one week. They were fed 
with a feed "CA-1" (containing iron in an amount of 31.5 mg/100 g, and 
manufactured and sold by Nippon Kurea K.K., Japan), and city water was 
given. During the preparatory breeding, weight increase was checked. Those 
rats having a body weight of about 160 g at the age of six weeks were 
subjected to the experiments. Through the period of experiments, the rats 
were bred in a metabolic cage. No feeding was done for 18 hours before 
administering a test sample, but distilled water was continued to be 
given. Each test sample prepared according to the above-mentioned 
procedures was administered to a group of five rats, directly into their 
stomach by means of stomach probe, in an amount of 1 ml/160 g body weight. 
Six hours after administering the test sample, feeding of feed "CA-1" was 
resumed. 
(3) Determining Radioactivity of In-vivo Sample: 
After administering each test sample, feces and urine were taken at 
intervals of hours to determine radioactivities thereof. 50 .mu.l of blood 
was taken by means of a heparin-treated capillary from the tail vein at 
intervals of hours, and subjected to centrifugation at 3,000 rpm for 5 
minutes to separate blood corpuscles from plasma. Radioactivities of the 
so obtained blood corpuscles and plasma were determined. Absorption of 
iron into the body and incorporation of the body-absorbed iron into 
hemoglobin are shown in Table 4. 
TABLE 4 
__________________________________________________________________________ 
(Comparison between organoiron(II)-containing wine, 
inorganic ferrous compound and inorganic ferric 
compound, with respect to absorbability into the 
living body and incorporation of iron into hemoglobin) 
Iron Dose A B B/A 
concentration 
(Fe .mu.g/ 
Absorb- 
Incorporation 
Utility of 
in sample 
kg .multidot. body 
Kind of 
ability 
into Hemoglobin 
absorbed 
(.mu.g/ml) 
weight) 
sample 
(%) (%) iron (%) 
__________________________________________________________________________ 
10 62.5 (a) 69.4 .+-. 1.4 
48.0 .+-. 1.8 
69.2 .+-. 1.5 
(b) 67.4 .+-. 3.6 
47.5 .+-. 1.8 
70.5 .+-. 2.5 
(c) 50.1 .+-. 4.2 
32.0 .+-. 6.2 
63.9 .+-. 2.6 
40 250 (a) 53.5 .+-. 1.7 
28.5 .+-. 2.3 
53.3 .+-. 2.5 
(b) 37.6 .+-. 4.0 
20.0 .+-. 1.9 
53.2 .+-. 4.0 
(c) 29.9 .+-. 6.9 
15.7 .+-. 4.2 
52.5 .+-. 4.5 
(d) 31.0 .+-. 3.5 
17.9 .+-. 3.2 
57.7 .+-. 3.5 
80 500 (a) 35.5 .+-. 4.6 
17.1 .+-. 2.3 
48.2 .+-. 4.5 
(b) 20.0 .+-. 4.0 
10.5 .+-. 1.9 
52.5 .+-. 10.5 
(c) 16.5 .+-. 3.1 
7.2 .+-. 2.0 
43.6 .+-. 3.1 
250 1500 (a) 23.5 .+-. 3.5 
7.2 .+-. 0.9 
30.6 .+-. 3.0 
(b) 9.7 .+-. 4.5 
3.8 .+-. 2.0 
39.2 .+-. 8.0 
(c) -- -- -- 
__________________________________________________________________________ 
Note 
A (absorbability) = 100 .times. (1 - amount of discharged .sup.59 
Fe/amount of total dose of .sup.59 Fe); and 
B (Incorporation into Hemoglobin): .sup.59 Fe content in the whole blood 
at the time of 168 hours after administration of sample. 
Each figure represents a mean value .+-. standard error. 
As is apparent from Table 4, when the iron concentration in administered 
sample of the present composition is as low as about 1 mg/liter or less, 
there is not a remarkable difference in absorbability into a living body 
between the organic iron complex according to the present invention and 
the inorganic iron salts. This is assumed to be attributed to the fact 
that such an iron concentration as low as about 1 mg/liter or less is 
within such a range that the reducing capacity of the living body itself 
is sufficient therefor. But, when the iron concentration is relatively 
high, the antianaemic composition according to the present invention 
exhibits a remarkably improved absorbability into the living body as 
compared with FeSO.sub.4 and FeCl.sub.3. On the other hand, no significant 
difference in absorbability into the living body is seen between the 
mixture of concentrated ordinary wine and ferrous sulfate [i.e. (d) in 
Table 4] and ferrous sulfate per se [i.e. (c) in Table 1]. 
EXAMPLE 2 
Substantially the same procedures as in Example 1 were repeated except that 
a semi-synthetic culture medium of Table 2 was employed as the nutrient 
medium. The mixture was subjected to fermentation at 30.degree. C. for 15 
days. The cultured broth was subjected to removal of alcohol at 20.degree. 
C. under a reduced pressure of 10 mmHg to an extent that the alcohol 
concentration of the cultured broth was decreased to 0.3% by volume to 
obtain an antianaemic composition. The iron content of the composition was 
35 ppm. The organoiron(II) compound-containing composition was very stable 
and excellent in antianaemic activity. 
EXAMPLE 3 
Substantially the same procedures as in Example 1 were repeated except that 
a synthetic culture medium of Table 3 was employed as the nutrient medium. 
The mixture was subjected to fermentation at 30.degree. C. for 15 days. 
The cultured broth was subjected to removal of alcohol at 20.degree. C. 
under a reduced pressure of 10 mmHg to an extent that the alcohol 
concentration of the cultured broth was decreased to 0.7% by volume, 
followed by filtration to obtain a supernatant. The iron content of the 
supernatant was 33 ppm. The organoiron(II) compound-containing supernatant 
was very stable and excellent in antianaemic activity. 
Turning now to FIGURE, there are shown the relationships between the iron 
contents in the living body at its plasma, bone-marrow, erythrocyte and 
small intestine and the lapse of time after administration of the sample 
prepared from the supernatant obtained in Example 2 and containing 250 
.mu.g Fe/ml in a total dose of 250 .mu.g Fe/kg.multidot.body weight. The 
tests were carried out in substantially the same manner as in the 
above-mentioned Experiments, items 2) and 3) to determine the change in 
iron content in the living body at its plasma, bone-marrow, erythrocyte 
and small intestine with the lapse of time after the administration. As is 
clearly seen from FIGURE, first, the iron content in the plasma rapidly 
increases but, in turn, rapidly decreases to reach nearly zero about 168 
hours after the administration. Instead, second, the iron content in the 
bone-marrow increases and begins to decrease about 20 hours after the 
administration. Third, the iron content in the erythrocyte gradually 
increases to reach a plateau region (in which the iron content is larger 
than those observed with respect to the plasma and the bone-marrow) and 
the iron content in the erythrocyte which has once reached the plateau 
region does not change for about 50 days. The graphs suggest that the iron 
values absorbed in the plasma are transferred to the erythrocyte through 
the bone-marrow. The graphs also show that the organoiron(II) compound of 
the present antianaemic composition is very stably absorbed into the 
living body and that the incorporation of iron into hemoglobin is very 
large. Therefore, the times of dosage can advantageously be reduced.