NUTRITIVE FACTORS IN ANIMAL TISSUES. I. *

BY THOMAS B. OSBORNE AND LAFAYETTE B. MENDEL.

WITH THE COOPERATION OF EDNA L. FERRY AND ALFRED J. WAHEMAN.

(From the Laboratory of the Connecticut Agricultural Experiment Station and the Shefield Laboratory of Physiological Chemistry in

Yale University, New Haven.)

(Received for publication, October 30, 1917.)

Our early experience’ in attempts to feed white rats with artificial mixtures of purified foodstuffs led us to the conviction that something in addition to the proteins, fats, carbohydrates, and inorganic salts, which were then regarded as the only essentials of an adequate diet, was necessary for the normal nutrition of these animals. This was made evident by the rapid growth of young rats fed with mixtures containing a part of the carbohydrate and all of the inorganic salts in the form of our so called “protein- free ” milk, whereas those fed with chemically similar diets which lacked the small amount of the unknown constituents of milk grew little if at all. These observations coincided with earlier ones by Stepp2 (on mice) and those of Hopkins3 who had independently reached the same conclusion. Subsequent inves- tigations by ourselves and others have greatly extended the knowledge of the need of both growing and adult animals for those still chemically unknown substances, which are now generally designated as food hormones, or vitamines. Of these, two types

* The expenses of this investigation were shared by the Connecticut Agricultural Experiment Station and the Carnegie Institution of Wash- ington, D. C.

1 Osborne, T. B., and Mendel, L. B., Carnegie Znstitution of Washington, Publication No. 166, pts. i and ii, 1911.

* Stepp, W., Biochem. Z., 1909, xxii, 452; 2. Biol., 1911-12, lvii, 135. 8 Hopkins, F. G., J. Physiol., 1912, xliv, 425. 4 For a statement of the development of certain of our ideas on these

topics see Osborne and Mendel, Biochem. J., 1916, x, 534. 309

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310 Nutritive Factors in Animal Tissues. I

appear to exist: one soluble in fats, the other soluble in water. They have been shown to be present in the following animal and vegetable products.

Food Hormones.

Fat-soluble.

Butter fat.* Egg yolk fat7 Cod liver oil.* Beef fat.9 “Oleo-oil” margarines.gv 25 Pig kidney fat.107 1’ Maize kernel.“* 18. 20 Rye.” Wheat embryo.“* ‘3

‘L kernel.” Cotton seed.rls 20~ 27 Cabbage leaves.‘89 23 Clover “ 18, 23

Oat kernel.18 Soy beans.*O* *e Sunflower seed.21 Alfalfa leaves.*“, 23 Flax seeds.22, *a Hemp seed.22 Millet seeds.22* 23

Watersoluble.

Milk.3, 5 Rice.12 Wheat embryo.13’ 27 Cotton seed.14 Pancreas.r5 Maize kernel.16 Wheat “ 17 Oat “ I8 Kidney beans.19 Yeast.?” Soy beans.26 Maize embryo.?8 Peanut meal.?*

5 Osborne and Mendel, Caljlegie Institulion 0.r Washington, Publication No. 156, pt. ii, 1911; Science, 1911, xxxiv, 722; J. I3iol. Chem., 1912, xii, 473; 1912-13, xiii, 233.

B Osborne and Mendel, J. Viol. Chem., 1913, xv, 311; 1913-14, xvi, 423; 1914, xvii, 401; 1915, XX, 379. McCollum, E. V., and Davis, M., J. Biol. Chem., 1913, xv, 167.

7 Osborne and Mendel, J. Biol. Chem., 1914, xvii, 401; 1915, xx, 379. McCollum and Davis, J. Biol. Chem., 1913, xv, 167. MacArthur, C. G., and Luckett, C. L., J. Biol. Chem., 1915, xx, 161.

8 Osborne and Mendel, J. Biol. Chem., 1914, xvii, 401. hlcCollum and Davis, J. Biol. Chem., 1915, xx, 641.

s Osborne and Mendel, J. Biol. Chem., 1915, xx, 379. I0 McCoIlum and Davis, J. Biol. Chem., 1915, xx, 641. ** McCollum and Davis, J. Biol. Chem., 1915, xxi, 179. I2 McCollum and Davis, J. Biol. Chem., 1915, xxiii, 181. I8 McColIum, E. V., Simmonds, N., and Pits, W., J. Biol. Chem., 1916,

xxv, 105. I4 Richardson, A. E., and Green, H. S., J. Biol. Chem., 1916, xxv, 307;

1917, xxxi, 379.

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T. B. Osborne and L. B. Mendel

It is to be noted that in the above lists milk and pancreas are the only products of animal origin that have as yet been tested for the presence of the water-soluble vitamine. Indirect evidence that animal tissues may contain this hormone is afforded by studies of the protective, curative, or antineuritic properties of some of these in relation to experimental polyneuritis or beri- beri. That the substance which induces the remarkable recoveries which have been described in these cases is identical with the water-soluble hormone which is so essential for growth and maintenance is as yet merely a matter of conjecture.

Suzuki, Shimamura, and Odake2g reported that an alcoholic extract of horse meat was unable to prevent the death of mice fed on a diet of polished rice; and that dogs fed exclusively on polished rice and boiled horse meat became greatly emaciated and ill in 3 to 4 weeks. Cooper,3O who has reviewed the scanty but often suggestive earlier literature up to 1914, has compared the antineuritic properties of animal tissues of various sorts with respect to their relative potency in preventing or curing avian polyneuritis induced by feeding polished rice. The following excerpts from his protocols will serve to indicate the general results.

16 Eddy, W. H., J. Biol. Chem., 1916, xxvii, 113. 16 McCollum, Simmonds, and Pitz, J. Biol. Chem., 1916-17, xxviii, 153. 17 McCollum, Simmonds, and Pitz, J. Biol. Chem., 191G-17, xxviii, 211. ~8McCollum, Simmonds, and Pitz, J. Biol. Chem., 1917, xxix, 341. 19 McCollum, Simmonds, and Pitz, J. Biol. Chem., 1917, xxix, 521. 20 McCollum, Simmonds, and Pitz, Am. J. Physiol., 1916, xli, 361. 21 McCollum, Simmonds, and Pitz, Proc. Sot. Exp. Biol. and Med., 1916,

xiii, 129. 22 McCollum, Simmonds, and Pitz, J. Biol. Chem., 1917, xxx, 13. 23 McCollum, E. V., J. Am. Med. Assn., 1917, lxviii, 1379. 24 Osborne and Mendel, J. Biol. Chem., 1917, xxxi, 149. 26 Halliburton, W. D., and Drummond, J. C., J. Physiol., 1917, Ii, 235. 26 Osborne and Mendel, J. Biol. Chem.,l917 xxxii, 369. Daniels, A. L.,

and Nichols, N. B., J. Biol. Chem., 1917, xxxii, 91. p1 Osborne and Mendel, J. Biol. Chem., 1917, xxix, 289. 28 Osborne and Mendel, unpublished data. 29 Suzuki, U., Shimamura, T., and Odake, S., Biochem. Z., 1912, xliii, 89. *O Cooper, E. A., J. Hyg., 1912, xii, 436; 1914, xiv, 12.

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312 Nutritive Factors in Animal Tissues. I

Amount of Animal Products Necessary to Prevent Polyneuritis. According to Cooper.

Ox voluntary muscle. “ cardiac “ . “ cerebrum. “ cerebellum. “ liver..............

Cow’s milk.. . Sheep cerebrum _. . Fish voluntary muscle Egg yolk. . Cheese................

......

......

......

. . . . . .

......

......

...... .......

-

I

--

-

‘n ternIs of natural foodstuff.

Pm.

20 5 6

12 3

>35 8 to 15

>I0 3

>8

-

!

-

vm. 5.0 1.7 1.2 2.4 0.9

>3.5 1.6 to 3

>2 1.5

>5.6

In 1913 Funk31 made the following statement: In the animal kingdom unheated milk and egg yolk are rich in vitamines; these foods must supply the great need of vitamines on the part of young, growing organisms; furthermore these substances are found in meat, especially abundantly in heart muscle, in brain, and probably in most animal tissues. In a tabular summary he refers to meat juice, meat extract, and slightly roasted meat as containing vitamine; and to sterilized meat extract and soup meat as being devoid of vitamine. We assume that this refers to the antineuritic properties of the substances mentioned. It is unnecessary to offer a critique of the significance of the results of these earlier statements from the standpoint of the vitamine hypothesis of the present time, which takes into account all of the possible factors which may be involved in an inadequate diet.24’ 82 The problems of growth were not considered.

In view of the great importance of food hormones in the diet and the wide use of animal tissues of various kinds for food it has seemed desirable to undertake a comprehensive study of this question. Furthermore, it has seemed possible that in parts of

31 Funk, C., Miinch. med. Woch., 1913, 2614, “In der Tierwelt ist rohe Milch, das Eigelb reich an Vitaminen; diese Stoffe miissen den grossen Bedarf an Vitaminen bei jungen wachsenden Organismen decken; ferner befinden sich diese Substanzen im Fleisch, besonders reichlich im Herz- muskel, im Gehirn und wahrscheinlich in den meisten Tiergeweben.”

a2 McCollum, E. V., and Pits, W., J. Biol. Chem., 1917, xxxi, 229.

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T. B. Osborne and L. B. Mendel 313

the various animals which are now little or not at all used for human consumption there may bc some which are part.icularly rich in the water-soluble food hormone and that consequently these, if not suitable for human food, could at least, be more advantageously fed to growing domestic animals than is now the case.

The results of our experiments with beef muscle and pig liver which are described in this paper were of such a character that we are now actively engaged in extending this study to a greater variety of animal products. We have thus far investigated beef muscle, meat extract, the tissue residue from this meat extract, and dried pig liver, follotving t,he plan of our earlier studies with rats. The substance under investigation was fed as the solo source of protein and water-soluble vitamine in t,hc diet, combined with sukable carbohydrates, fat,s, and inorganic Salk, or merely as the sole source of water-soluble food hormono or vitamine in an otherwise adequate food mixture.

The “v/eat pouder” was prepared by grinding fresh lean rolmd of beef very fine, drying on pans in n current of hot. ::ir, thtan in an oven at 105”C., and grinding t,o a powder. ‘I’he “~nonf ~ztrwt” was prepared by grinding finely chopped lean round of beef through a Nixtnmal mill with nearly three times its lwight of distilled water. The t.llin pulp was then boiled for a few rninutcs over a free flame, filt,ered, and t,lle extract concentrated over a stcnm hath or in a current of hot air, nearly dried in a vacuum dcsiccn- tor and then in an oven at 105”C.s* In t,his may 17.3 per cent of meat extract in terms of the total meat solids-each calculated on a wnter- free basis--was obtained. This is a yield considcrnbly larger than that usually reported for the water-soluble non-coagulal~lc components of meat; it is doubtless due to t.he extreme degree of comminution obtained by grinding with much wai,(lr in a suitable mill. Before mixing with the other ingredients in the food the meat extract was dissolved in a little wat.cr, mixed with a qantity of cornstarch and dried on pans in a cur- rent of hot air. The “nzeat wsidzre” was prepared by pressing the solid portion remaining from the filbation of the extract, in a hydraulic press, reextracting it with water, drying in a current of hot air, and finally in an oven at 105°C. The dried liver was prepared by removing the 1:trgcr blood vessels from the fresh liver as completely as possible, drying t.he material in a current of hot air, snd finally in an ovrn at 105°C.

33 This method of drving is essentially the same as WC have employed in making “protein-f&z milk.” Our extensive eq)cricnc:e wit,h this ma- terial has given us no reason to believe that, the vitamines are damaged to any great extent by the heating.

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314 Nutritive Factors in Animal Tissues. I

The results of the nutrition cxperiment,s are shown in graphic form in the accompanying ch&s. Both the meat powder and the meat extract seem to be deficient in the water-soluble food hor- mone. When yeast, “protein-free milk, ” corn germ, or wheat embryo-all tested sources of the water-soluble food hormonc- were added to the diet, a rapid response in growth was made by the rats; whereas without them nut,ritive failure invariably ensued. E’rom this it, is evident that both the meat powder and the meat residue arc suittable as sources of protein in the diet, when the other essential ingredients are present in sufficient amounts. This needs emphasis in view of the fact that, meat residues such as remain after the preparation of soups from muscle tissue are oft’cn discarded as inferior food product’s, The pro- t,eins which they still contain are entirely adequat.e for nutrition in growth.

The meat extract apparently contains at least a small amount of the wat,er-soluble food hormone; for the rats on the edestin- meat’ extract food have grown somewhat bet,ter than t,hc: majority of our animals which have been entirely deprived of this vitamine. The qunmity of meat extract in t,his food, however, is equivalent to almost twice as much as that in the meat which the rats on the meat powder foods received.

In striking contrast to these results obmined wit,h the muscle tissue and its extract arc those furnished by the experiments with the dried pig liver. They demonstrate that this organ contains both adequat,e protein and a large proportion of water-soluble vitamine. These results parallel the findings of Cooper in respect, t,o the relative nntineuritic properties of muscle and liver, and also agree with those of Etldy,15 which “seemed to prove con- clusively t,hat the water-soluble portion of the acloholic extract of pancreas contains a subst.ance that is capable of inducing marked increase in growth. ” It is to be noted that both liver and pancreas, in contrast to muscle tissue, are exceptionally rich in glandular cells.

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CHARTS.

315

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316 Nutritive Factors in Animal Tissues. I

EXPER MENTS WITH 1 MEAT POWDEF

T-mAsT D/E75 W/i-N YEAST --..-..

CHART I.

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T. B. Osborne and L. H. 1Iendel 317

CHART I. Showing the failure of young rats to grow on a diet of meal powder, starch, fats, and a salt mixture (Periods 1, 2, and 4) except when a small amount of yeast was furnished in addition (Period 3). I’hc addi- t,ion of butter fat in Period 2 in place of lard supplied the fat-soluble food hormone without averting the nutritive decline. The quantity of yeast added in Period 3, during ~vhich t,he animals grew very rapidly, amounted to only 0.2 gm. per day. This n-as fed sepnrntcly. The nit,hdrawal of yeast, in Period 4 was soon followed by nutritive disaster although t,his occurred somewhat less speedily than in the cxperimcnts with ?rrenl rc.sidrLe, Chart II, Period 3, doubtless owing to t,he small quantity of water-soluble food hormone present in the whole wzrnt ~o~rlcr but entirely lacking in the ?rLCOl residue. The highly sucocssful growth \vhcn all of the other essentials were present (Period 31 shows that the meat proteins per se arc entirely adequate for nutrition in growth.

The composition of the foods in t,he different periods was as follows:

.\Ient pan-dcr ...... .........

-~ -iGpFjqT-

... ... d Salt mixture ....................... 4 4 4 Starch. ............................ 61 61 61 Lard .............................. 15 Butter fat ......................... 13 15 Dried yeast ....................... 0 2 gni. per diem.

Sitrogen content .................. 2.4 2.4 2.G

The salt mixture used in all of these espcrimcnts had the following composition:

l7n. gm. ChCO~. ..................... 134.8 Citric acid + Hz0 ....... 111 .l MgC03. .................... 2-1.2 Fe citrate 1; Hz0 ........ 6.34 Sa*CO 3 .. .................. 34.2 ICI. ...................... 0.020 I&CO3 ...................... 141.3 .\InS04. .................. 0.079 H3P04 ..................... 103.2 NnF. .................... 0.248 HCl.. ...................... 53.4 &Xl* (,So,)z. ............. 0.0245 H,SOd ........... ........... 0.2

The chemicals used were analyzed and nllo\~:tnrc \vns mndc for moisture, etc. The ncids were mixed and the carbonates and ferric citrate added to them. The traces of ICI, MnSOI, NnF, and I<?~\12 (SO,)2 were added as

solutions of known concentrations. The final resulting mixtjure was evaporated to dryness in’a current of air at OfkIOO”C., and ground to a fine powder.

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318 Nutritive Factors in Animal Tissues. I

100

80

60

&ART II.

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T. B. Osborne and L. B. Mendel 319

CHART IT. Showing experiments comparable with those in Chart I except that meat residue from which the extractives had been removed was used in place of the meat powder. Hem, too, there was a failure of nutrition except when dried yeast (amounting to 2 per cent of the food mixture and equal to about 0.2 gm. per day) was present in the ration (Period 2). As csplained under Chart I, the faiIurc on the foods n-ithout added sources of water-soluble food hormone are more prompt in these meat residue experiments than in the trials with the m‘eat powder. The

proteins of the meat residue are thus proved to be entirely adequate for growth n-hen all other cssentiak are furnished in the ration.

The composition of the food mixtures is as follows:

-

Meat residue ..................... Salt mixture*. ..................... Starch ............................ Butter fat ........................ Lard ............................. Dried yeast ...................... Nitrogen aontent .................

Period 1. Period 2. Period 3.

per cent per cent

18.5 18.5

4.0 4.0 53.5 51.5

18.0 24.0 6.0

2.0 2.8 3.0

per cent

18.5

4.0 53.5 18.0

6.0

2.8

* For composition of salt mixture see legend for Chart I.

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320 Nutritive Factors in Animal Tissues. I

CHART III.

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T. B. Osborne and L. B. Mendel 321

CIIART III. Showing t,he nutritive decline owing to the lack of wat,er- solul)le food hormone in the meat residue (Period 1) and the prompt response in growth when various sources of the wnttlr-soluble food hor- mone such as protein-free milk, corn gcrnis, lvhe:it embryo, and liver c-erc added in subseqiicnt perioA. The ~~rrs~ncc of 3 per cent of meat ext,ract (cquivnlent to t,he amount origimtlly prrscnt in the meat) in tht diet, of Rats ‘1362, 4357, 4347 (Period 1) evidently did not furnish sufficient of tlrcb hormone to avert, the declinc~. Even 5 per cent of mc:it oxtrnct in the diet was insufficient when tcstcd with Rats 43350 and 4354 (Period 2). The results afford striking examples of the importance of properly com- bining ordinary foodstuffs so that sufficient quantities of all of the dietary

Rnts I Itats Rat

4350 434i 4362 4354 4357 4381

-!, “P 1 P

Meat residue hIcat cstrnct _. _. Corn grrms Wheat embryo Dric>d liver _. ProtAn-free milk Salt, niisture Starch. Butter fat.. 1 1 I

Lard

5

4

53

-8

6

2.3

Period 1.

essen(ials are furnished. The composition of t,hc food mixtures was :rs follows:

i’rriod !?

28.0

Nitrogen content 2.4 2 8 I

48.5 29.5

IS.0 18 0 GO G 0

3.3 3 0

-- Period

3.

Rats

4350 4354

40 48.5

J 18.0

G.0

3.3

5.0

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322 Nutritive Factors in Animal Tissues. I

CHART IV. Experiments in which 5 per cent of meat extract ~vas used as the sole source of food hormones. The growth of these rats was de- cidedly better than experience with vitamine-free diets of comparable compositioh would lead us to expect,; but the normal rate of growth has not been sustained. The failure at this stage was not due to the lack of the fat-soluble hormone; for growth was not improved by the addi- tion of butter fat in Period 2. Furthermore inasmuch as rats recorded in Chart III failed to grow even with an abundance of butter fat in the diet,, when meat extract served as the source of the water-soluble’hormone, but responded promptly to other sources of the latt,er, the failure of the rats on this chart cannot be attributed to a lack of fat-soluble hormone. We, therefore, conclude that the meat extrnct contains at best only a small proportion of the water-soluble food hormone.

The composition of the food mixtures was as follows:

Period 1. Period 2.

per cent per cent Edestin.................................. 18 18

Salt mixture............................. 4 4 Meat extract............................ 5 5 Starch................................... 50 50 Lard..................................... 23 5 Butter fat................................ 18 Nitrogen content.. . 3.65 3.65

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T. B. Osborne and L. B. Mendel 323

CHART V. Showing the high efficiency of dried pig liver as the source of bot.h protein and water-soluble food hormone for nutrition during growth. The contrast between the success of the experiments with liver and those of muscle products shown in the preceding charts is most striking.

The composition of the food mixtures was as follows:

I Rats I Rats

L)ried liver.. Salt mixture*. Starch . . . . Butter fat.. Lard. . . . . Nitrogen content..

. . . . .

23 4

50 I 18

l.4

! 4371 4369 4368

- per ceni

23 4

50

23 2.4

* For composition of salt mixture see legend for Chart 1.

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CHART 6. Lots 837 and 839 make clear the beneficial effects of having B liberal supply of the unidentified A and B in the diet. All the animals were undersized in both lots but in Lot 837 with 3 per cent of butter fat (fat-soluble A) two females failed to have any young, while in Lot 839 which made no better growth two females produced one litter each and a third three litters. None of these lived beyond a few days. The higher plane of butter fat improved the vigor of the rats as shown in their ability to produce young. They needed still more of the factors A and B to enable them to rear their young (16).

In the first paper of this series we pointed out that in our studies of mixtures of maize and bean proteins an amount of pro- tein just sufficient to support growth at about the normal rate does not induce well-being in the degree that a moderate excess of protein over the actual requirement does.

Perhaps the data presented in this paper are not sufficient to justify a definite conclusion, but it appears that the young are more tolerant for a time than the adult of diets relatively deficient in the fat-soluble A and water-soluble B. The lack of uni- formity of animals of apparent good vitality on such diets as we are describing emphasizes the probability that the vigor of the young may explain the results.

These data support the idea that for best results the diet should not approach the minimum requirements of any dietary factor.

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CIUI.T LOT (00 -__

115

x5

35

GM

195

I!55

115

75

35

CHART 4. Lot 798 shows that a considerable amount of growth in young rats is possible on the amount of the dietary A and B in 1 per cent of butter fat and wheat germ respectively. It is a surprising fact that all suffered a period of suspension of growth at the beginning of the experiment, followed by a period of subnormal growth. This is not due to error in making up the ration, for it was made up anew at intervals of about 3 weeks during the entire experiment. Furthermore, the animals did not begin to grow at the same time.

Lots 800 and 885 illustrate the amount of growth which young rats are capable of making on a mixture in which 1 per cent of wheat germ furnishes the B and 2 and 3 per cent of butter fat the A. The young tolerated this diet better than one would expect from the behavior of adults on the same mixture (Chart 2, Lots 608 and 664).

In Period 2 of Lot 885 an alcoholic extract of wheat germ was added to the diet to suppIy more of the factor B. AIthough somewhat delayed there was a noticeable response in all individuals. One rat suffered two attacks of polyneuritis and was each time relieved within 48 hours by small doses of a water extract of wheat germ administered with a medicine dropper, since it was unable to stand.

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CHART 2. This chart illustrates the behavior of adult rats when fed purified foodstuffs as in Chart 1, but with the water- soluble B supplied by 1 per cent of wheat germ and the fat-soluble A by 1, 2, and 3 per cent of butter fat respectively in Lots 662, 664, and 608.

On the first of these (Lot 662) none of the rats were able to maintain their weight, but in one case a female lived 400 days. Two individuals were unable to tolerate the limitations of the diet and suffered rapid decline.

Lot 664 which received 1 per cent of wheat germ and 2 per cent of butter fat practically maintained their weight in all in- stances. One individual (male at top) lived 143 months with a considerable gain in weight followed by decline. The other three, while not losing weight, had their lives greatly shortened bJ- the dietary deficiencies. One which produced a litter of young, all of which died soon after birth, later suffered typical polyneuritis.

In Lot 608 which received its unidentified dietary factors from 1 per cent of wheat germ and 3 per cent of butter fat did not contain a single individual among the nine animals in the group which was able to tolerate the diet without distinct loss of vital- ity, and greatly shortened span of life. The danger of degenerative changes is very great when the content of the dietary essen- tials A or B is reduced to near the minimal requirement.

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Alfred J. WakemanWith the cooperation of Edna L. Ferry and

Thomas B. Osborne, Lafayette B. Mendel andTISSUES. I

NUTRITIVE FACTORS IN ANIMAL

1917, 32:309-323.J. Biol. Chem. 

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