we pointed out that the nutritional anemia induced in rabbits by a whole milk

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IRON IN NUTRITION.
1V. NUTRITIONAL ANEMIA ON WHOLE MILK DIETS AND ITS
CORRECTION WITH THE ASH OF CERTAIN PLANT AND
ANlMAL TISSUES OR WITH SOLUBLE IRON SALTS.*
BYE. B. HART, C. A. ELVEHJEM, J. WADDELL, AND R. C. HERRIN.
(From the Department of Agricultural Chemistry, University of
Wisconsin, Madison.)
(Received for publication, December 21, 1926.)
In an earlier publication (1) we pointed out that the nutritional
anemia induced in rabbits by a whole milk (cow) diet could not be
corrected by the addition of inorganic iron (Fez03) but that such an
anemia could be corrected when in addition to the Fez03 there was
added to the diet either fresh cabbage or a cold 95 per cent alco-
holic extract of desiccated cabbage or an alcoholic extract of corn-
meal. These extracts were practically free from iron. A chloro-
phyll preparation free from iron fed in the presence of added ferric
oxide was also an effective supplement to a whole milk diet as a
prophylactic for anemia.
From these experiments we deduced the conclusion that some
organic nucleus ‘necessary for hemoglobin building was absent or
deficient in whole milk. This deduction was in harmony with the
generally accepted theory (but for which there was no experimen-
tal proof) that there may be a deficiency in whole milk of some
nucleus constructed from a multiple of pyrrole groups and which
is necessary for hemoglobin building. It was this idea which led
us to add a preparation of chlorophyll to whole milk supplemented
with FenOa and which gave us positive results in respect to anemia
prevention.
As our work on this problem progressed, we had accumulated
dat,a showing that not only were the corn grain and fresh cabbage
* Published with the permission of the Director of the Wisconsin Agri-
cultural Experiment station.
299
This is an Open Access article under the CC BY license.
300 Iron in Nutrition. IV
effective agents in the prevention of anemia in the rabbit con-
fined to a whole milk (cow) Fez03 diet, but that lettuce was also
especially effective as a preventive of anemia. We had included
lettuce in our category of supplements because Evans (2) and as-
sociates had used it with marked success as a source of vitamin E
and we were not sure at that time but that there might be some
direct relation between vitamin E and our problem of milk defi-
ciency and nutritional anemia.
TABLE I.
Potency of Fresh Lettuce in the Cure of Nutritional Anemia.
-
Rt-zTit HISlO-
Data Diet. globin. I Zrythrocytes Weight.
- -- -- --
198.5 per e.mm. om.
94 June 12 Whole milk only. 365
“ 25 8.9 4,500,000 640
July 1 5.9 3,550,ooo 780
“ 2 Added Fe203 (0.015 gm.
daily) + 50 gm. fresh
lettuce daily.
“ 10 8.5 6,300,OOO 990
“ 16 9.8 6,275,OOO 1170
“ 17 Discontinued; in perfect
condition.
- - -
We tried to feed dried lettuce in order to demonstrate that drying
would not injure its potency, but the dried material seemed to be very
unpalatable and the rabbits would not eat the preparation.
In Table I is shown a typical record of the cure of an anemia
induced in the rabbit by a whole milk-Fe203 diet, by the use of
fresh lettuce. The amount of hemoglobin and the erythrocyte
count increased almost twofold in a period of 2 weeks.
Before making further attempts to separate the active principle
of these plant materials functioning as cures or preventives of
anemia on a whole milk-Fe203 diet, it was decided to determine
whether the ash of these materials would be effective. We had
tentatively assumed that the preventive principle was of an organic
character. Our work up to the present has been mainly with
lettuce ash although the ash of other materials has also been tried.
Hart, Elvehjem, Waddell, and Herrin 301
So successful have been the cures or preventions of nutritional
anemia induced by a whole milk-Fe203 diet with the ash of certain
materials, that we feel certain that the deficiency of cow’s milk
in respect to its anemia-producing properties is not an organic
principle but something of inorganic character resident in the ash
of certain plant and animal tissues. These ashes of plant and ani-
mal materials were prepared in an electric mufRe operating at a
temperature of 650-750°C. and for a period of 20 to 30 minutes and
were devoid of carbonaceous materials. On occasions the ashes
were prepared in gas-heated furnaces at temperatures of a cherry-
red. The use of such temperatures must make it certain that we
are dealing in these experiments with inorganic materials and not
with organic substances.
EXPERIMENTAL.
For all the experiments to be described in this paper rabbits
have been used. We have also used chicks confined to a special
diet as checks on the work with the rabbit, but the ration used
and the results secured with chicks will be described in a
future publication. The young rabbits were always weaned from
the doe at 4 weeks of age and placed on the experimental diet.
The stock ration fed the does during the period of gestation
consisted of 100 parts of rolled oats, 10 parts of wheat bran, 1 part
of common salt, and whole milk ad libitum. Green materials
(cabbage, green grasses, or carrots) were given the mother until
the young rabbits were about 3 weeks old, but were withheld during
the last week of the suckling period. The experimental animals
were confined individually in cages fitted with $ inch mesh screen
bottoms to prevent feces consumption, and were weighed regu-
larly throughout the experiment. They usually weighed 300 to
400 gm. when placed on the experiment. In some cases we have
periodically irradiated the experimental animal with ultra-violet
light for the purpose of protecting against rickets. We have found
that this irradiation had no influence in protecting the animal
against an anemia on a whole milk diet. The practice of irradiating
our experimental animals has not been general because on the diet
used, there has been no direct evidence that our animals suffered
from rickens. Sodium citrate, used in our earlier experiments to
Iron in Nutrition. IV
prevent the formation in the stomach of large curds and possible
gastritis, has not been used in our later experiments. We have
found it unnecessary. Our experimental colony has suffered some
from intestinal diarrheas terminating in death, but the records
presented in this paper are from animals free from such disturb-
ances. Hemoglobin was estimated by the Fleischl-Miescher
hemoglobinometer and expressed as gm. per 100 cc. of blood.
A discussion setting forth the desirability of expressing the hemo-
globin content of blood by a uniform standard will be found in a
paper by Elvehjem and Waddell (3). Erythrocytes were deter-
mined in an American Standard hemocytometer with Levy count-
ing chamber.
The young rabbits 4 weeks old were given whole milk ad libitum
until the gm. of hemoglobin per 100 cc. of blood had decreased to
4.7 to 5.8 and the erythrocyte count to 3.5 to 4.5 millions. This
generally took a period of from 5 to 6 weeks. At this time the
milk was supplemented daily with 0.015 gm. of Fe20a and the
lettuce ash. The ash from 5 gm. of ground dried leaf lettuce was
triturated with the Fez03 in a small quantity of milk and was fed
each morning. This insured a more complete consumption of the
supplement. The animals were fed individually. During the re-
mainder of the day the rabbits were allowed an excess of milk.
Table II contains the records of two of the rabbits and is typical
of the improvement noted after feeding the lettuce ash to anemic
rabbits. Rabbit 180 after showing definite anemic symptoms
was fed lettuce ash plus FezO$ directly. Rabbit 164 upon show-
ing similar symptoms was fed Fe203 alone for a period of 19 days to
again show that Fez03 fed as such was ineffective in improving the
blood stream. Then the lettuce ash was added to the diet. One
may readily see from the table that as soon as the ash of lettuce
was added there was a steady improvement in the blood stream
and after a period of approximately 30 days the amount .of hemo-
globin and erythrocyte count was normal. After the blood stream
became normal, it remained so, provided the ash of lettuce was fed.
R.abbit 180 was maintained in perfect condition for 118 days and
Rabbit 165 for a period of 111 days. Although discontinued at
1 The Fez03 used was the same material used in all of our work and was a
Baker and Adamson product marked C.P.
Hart, Elvehjem, Waddell, and Herrin 303
TABLE II.
Potency 0f . Lettuce Ash in the Cure of Nutritional Anemia.
Date. Diet. gHlo.Cb?inK. UJ- Erytbrocytes Weight.
1966 per c.mm. gm.
act. 5 Whole milk only. 425
“ 16 7.2 4,600,OOO 540
Lt 22 5.9 4,050,000 660
“ 23 Added Fez08 (0.015 gm.
daily) + ash of 5 gm.
dried lettuce.
“ 29 6.3 3,950,ooo 725
Nov. 6 7.4 4,820,OOO 770
“ 12 9.3 4,900,000 870
“ 24 9.8 5,150,000 1150
Dec. 9 10 .o 5,970,ooo 1200
“ 29 12.3 6,650,OOO 1065
19.26
Jan. 6 11.0 6,170,OOO 1300
“ 13 11.5 6,700,OOO 1385
“ 20 11.7 6,200,OOO 1510
“ 29 11.5 6,350,OOO 1560
Feb. 10 11 .o 6,250,OOO 1790
“ 18 11.9 6,450,OOO 1850
19%
Sept. 29 Whole milk only. 590
Oct. 9 8.1 5,250,OOO 660
“ 20 7.0 5,300,000 840
‘I 26 7.6 5,200,OOO 850
Nov. 5 7.6 5,100,000 860
“ 8 6.3 3,700,000 860
“ 18 Added Fez03 (0.015 gm.
daily).
I1 27 5.1 3,100,000 975
Dec. 2 5.1 2,770,OOO 1020
“ 7 5.1 2,920,ooo 1020
‘I 8 Added Fe203 (0.015 gm.
daily) + ash of 5 gm.
dried lettuce.
“ 14 7.2 3,520,OOO 1040
(( 22 7.6 4,000,000 1160
“ 29 7.8 4,200,OOO 1225
304 Iron in Nutrition. IV
TABLE II-conduded. - - -
RFFt Date. HgleoRbiIno.- 1X rythrocytes Weight.
_- - - -_
g?n. per
1996 100 cc. per c.mm. 8”.
blood
164 Jan. 6 8.3 4,600,OOO 1335
“ 13 9.1 5,150,000 1460
“ 20 11.1 6,070,OOO 1470
“ 29 10.4 5,800,000 1625
Feb. 10 10.4 5,700,000 1900
“ 18 10.2 5,800,000 2060
Mar. I 8.5 5,500,000 2250
“ 17 9.8 5,900,000 2310
‘( 29 10.2 6,000,OOO 2450
that time there is no reason to believe that the normal condition
of their blood stream would not have been maintained indefinitely.
Although similar results were obtained with several other rab-
bits, the rapid improvement of the blood stream and the continued
maintenance of their good health produced merely by the addi-
tion of the inorganic elements in the lettuce ash with the Fe&&
was almost unbelievable. We were slow in being convinced that
this was true since our earlier results and those of several other
investigators had led to the conclusion that the factor limiting
hemoglobin building on a milk diet must be of organic nature.
Therefore we immediately started further experiments in order to
substantiate our data. More animals were used and greater pre-
cautions taken to minimize the role of possible genetic factors
entering into the experiment.
An entire litter of young rabbits at weaning time was placed
directly on the diet plus the various supplements. The preventive
type of experiment was used. The litter consisted of eight animals.
Each animal was given daily the whole milk plus 0.015 gm. of
Fez03. Two rabbits were given no further addition, two were
given the ash of 5 gm. of dried lettuce, two the ash of 5 gm. of
dried cabbage, and two the ash of 5 gm. of yellow corn. This gave
us an opportunity to compare rabbits from the same lit.ter, all of
which had received the same preexperimental feeding. The ex-
periment also made possible a comparison of the potency of the
ash of lettuce, cabbage, and corn on an equal basis. Two such
Hart, Elvehjem, Waddell, and Herrin 305
series were started at different times and the animals in each ex-
periment represented a single litter. The results of the entire
experiment were exceedingly uniform. The data obtained from
the two rabbits in each group and the two groups in each series
were so comparable that we present only the data from one rab-
bit in each group. These results are shown in Table III.
Again it was found that the ash of lettuce was exceedingly
beneficial in the prevention of nutritional anemia and the main-
tenance of a normal blood stream in a rabbit on a whole milk-
Fe203 diet. Rabbit 301 on the basal diet survived only 148 days,
during which period the average amount of hemoglobin was 8.0
FIG. 1. Rabbit 302, reared on a ration of whole milk-FepOa plus the daily
administration of the ash of 5 gm. of dried lettuce. Appears to be a perfect
specimen. Hemoglobin normal and erythrocytes per c.mm. about 5,700,OOO.
Started on the ration at 450 gm., and after 9 months on this ration weighed
2400 gm .
and the erythrocyte count 3,810,OOO. Rabbit 302 receiving in
addition to its whole milk-Fe203 diet the ash of 5 gm. of dried
lettuce made good growth and maintained a normal blood stream
over 200 days or 7 months and at this writing is continuing its
growth. The average gm. of hemoglobin per 100 cc. of blood dur-
ing the period was 10.4 and the erythrocyte count 5,950,OOO.
The rabbit appears normal in every respect. Fig. 1 is a photo-
graph of this animal taken September 13,1926. The results seem
to us exceedingly remarkable. This animal received no roughage,
TABLE III.
-
lhtc. Iht. Erythrocytcs. Weight.
yw per
lHZ6 100 cc. ym.
blood
301 1”cl). 17 \Vholc milk + Vc,Os 530
(0.015 gm. daily).
“ “7 s.7 4,700,000 GGO
I\lar. 10 7.0 3,000,000 980
“ l!) 8.7 4,100,000 1090
“ 26 x5 4,600,OOO 1190
:\I”‘. 5 7.G 3,850,OOO 910
“ 16 7.2 3,iOO,OOO 860
‘I “-1‘( 5 5 'L,GOO,OOO 950
Alay 3 72 3,800,000 1100
“ 12 4,s 2,400,000 1200
“ “4 i.G 4,000,000 1360
JUllC s 9 .3 4,800,000 1540
(( L’, s.5 4,300,000 li50
July 5 7.4 4,~00,000 1)520
“ 15 G.8 3,000,000 1750
July ‘24 Dead.
‘I’ypical record on \vhole rnilk-l”c,08-lettuce ash diet.
302 Pcl,. 17 \Yhole milk + FelOy 4i0
(0.015 gm. daily) +
ash of 5 gm. dried
lettuce.
(6 "7 10.0 6,200,OOO G80
hlar. 10 0.3 5,400,000 1000
“ 19 S.9 5,600,OOO 11'70
“ 36 10.6 6,200,OOO 1320
i\Pl'. 6 10.0 6,400,OOO 1420
“ 15 10.4 5,800,000 1460
“ 23 10.8 6,500,OOO 1560
May 3 12.1 7,200,OOO 1650
“ 12 12.1 7,400,000 1670
“ 3I 4 10 2 5,700,000 1730
June 8 10.0 5,600,OOO 1800
(I 21 10.4 5,700,000 2020
July 5 10.4 5,400,000 2200
“ 15 10 2 5,900,000 2310
“ 30 s.9 5,500,000 2280
Aug. 17 10.0 5,700,000 2450
“ 30 10 2 5,600,000 2510
Sept. 14 10.4 5,400,000 2520
Oct. 4 0.3 5,500,000 2520
“ 19 98 5,500,000 2400
Nov. 3 10.2 5,700,000 2390
306
Hart, Elvehjem, Waddell, and Herrin 307
TABLE III-Concluded.
RF2it Date. Diet. Erythrocytes. Weight.
Typical record on whole milk-FeeOs-cabbal ash diet.
gm. per
1966 100 cc. per c.mm.
blood
305 Feb. 17 Whole milk + Fez03 470
(0.015 gm. daily) +
ash of 5 gm. dried
cabbage.
“ 27 8.3 4,400,000 640
Mar. 10 8.9 4,700,000 840
‘( 19 9.6 5,800,OOO 990
“ 26 8.3 5,000,000 1170
Apr. 5 10.2 5,700,000 1300
“ 15 10.6 6,000,OOO 1460
“
23 10.6 6,200,OOO 1770
May 3 9.1 5,800,OOO 1900
“ 12 9.6 5,600,OOO 1900
(‘ 24 11.1 5,800,OOO 1880
June 8 10.0 5,600,OOO 1980
“ 21 9.3 5,400,000 2070
July 5 9.3 5400,000 2170
“ 15 9.8 5,800,OOO 2370
“ 30 10.0 6,000,OOO 2400
Aug. 17 9.8 5,500,000 2370
“ 30 8.5 4,500,000 2430
Sept. 14 10.0 5,600,OOO 2440
Typical record on whole milk-Fe203-corn grain ash diet.
295 Jan. 22 Whole milk + Fe203
(0.015 gm. daily) +
ash of 5 gm. corn.
Feb. 3 8.5 5,825,OOO 570
“ 10 8.9 6,000,OOO 600
“ 18 8.1 4,700,000 590
” 27 6.6 4,500,000 640
Mar. 9 6.8 4,600,OOO 810
“ 26 6.3 3,200,OOO 830
Apr. 3 6.1 3,100,000 830
“ 13 6.6 3,500,000 930
May 3 7.8 4,200,OOO 960
“ 18 7.0 3,800,OOO 1050
May 26 Dead.
308 Iron in Nutrition. IV
having been kept on a wire screen, and yet from all outward ap-
pearances, such as sleekness of coat, appetite, etc., appeared as a
normal specimen. Reproduction has not as yet been studied.
The ash of 5 gm. of cabbage was nearly as effective as that of
lettuce. This is shown in the record of Rabbit 305 (Table III).
Although the amount of hemoglobin and erythrocyte count was
TABLE IV.
Typical Record on Whole Milk-Fe203 + Ash of10 Gm. of Dried
Cabbage Diet.
Date. Diet. gIIloebmino.- Zrythrocytos Weight.
gm. per
f9M 100 cc. per c.mna. om.
blood
314 Mar. 9 Whole milk + FezOx 580
(0.015 gm. daily) +
ash of 10 gm. dried
cabbage.
“
20 7.2 4,300,000 660
“ 29 11.1 6,500,OOO 860
Apr. 15 10.6 6,000,OOO 980
“ 23 11.3 7,000,000 1180
May 3 10.6 6,500,OOO 1400
“ 12 9.6 5,900,000 1610
“ 24 8.7 4,700,000 1700
June 8 10.9 6,000,OOO 1950
“ 21 10.6 6,400,OOO 2090
July 5 10.4 6,100,OOO 2120
“ 15 10.0 5,700,000 2230
“ 30 10.2 6,100,OOO 2170
Aug. 17 10.6 5,800,OOO 2270
Sept. 14 9.3 5,100,000 2370
Oct. 4 9.1 5,000,000 2450
“ 19 10.6 5,900,000 2340
Nov. 3 9.8 5,700,000 2290
-
slightly lower for this animal than for Rabbit 302, it is still in our
colony, continues to grow, and appears to be normal.
Table IV shows the results secured with Rabbit 314 which was
one of the individuals in the later series. This rabbit received the
ash of 10 instead of 5 gm. of dried cabbage. It was found that, on
this higher level of feeding the amount of hemoglobin and the