GROWTH OF RATS ON “FAT-FREE” DIETS.

BY AVA JOSEPHINE McAMIS,* WILLIAM E. ANDERSON, AND

LAFAYETTE B. MENDEL.

(From the Laboratory of Physiological Chemistry, Yale University, New Haven.)

(Received for publication, February 19, 1929.)

In a series of studies on The Relation of Diet to the Quality of Fat Produced in the Animal Body, it was desirable to ascertain if possible, the composition of the fat yielded by an animal grown under a dietary regime in which there was no ingested fat. Ob- viously tissue fat formed under such conditions represents a product synthesized in the organism from precursors other than dietary fat. This product might be designated as “physiological” fat in contrast with exogenous fat transferred more or less un- changed from the food to the tissues. Accordingly, in order that such “synthetic” fat might be available for analysis, albino rats were grown on a ration practically devoid of fat. Under the conditions outlined, the growing of animals presented certain outstanding difficulties-notably, the necessity of feeding the so called fat-soluble vitamins without their commonly accompany- ing fat vehicles. The present communication refers to the meth- ods employed and the degree of success attained in surmounting the difficulties encountered.

The Experiments.

Experimental Animals.-With the exception of three females, as indicated on the charts, male albino rats taken at weaning, when approximately 21 days of age and weighing from 35 to 45

gm., were selected from our laboratory stock animals.’ With these rats-which were kept individually, some in cylindrical

* Holder of the Alpha Xi Delta Fellowship awarded by the American

Association of University Women (1928-29). 1 The laboratory stock animals were grown at that time on the Sherman

Diet B, modified (Smith, A. H., and Bing, F. C., J. Nutr., 1, 179 (1928)).

247

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248 Growth on “Fat-Free” Diets

false bottom wire cages and others in cages, consisting largely of glass, specially designed to facilitate cleaning-extensive precau- tions were taken to minimize contamination by fatty materials. In some cases as indicated on the charts, food was withheld during the 2 days immediately after weaning, before giving the experi- mental diet, in order that a partial depletion of the stored fat might ensue, thereby lessening its influence on the later resulting “synthetic” fat. In order to exclude, as much as possible, varia- tions due to individual litters, the rats of a given litter were fed different dietary supplements.

All animals were weighed twice weekly. Experimental Diets.-The selected diets, consisting of a “basal”

diet, fed ad libitum, and “accessories,” quantitatively supplied daily apart from the “basal” food, had the following composition :

Partially Purified. “Fat-Free” (and Modijications).

“Basal” Diet.

1. 18 per cent casein, commercial. 2. 77 “ “ sucrose. 3. 5 “ “ salt mixture.

1. 18 per cent casein, purified. 2. 77 ‘< “ sucrose. 3. 5 “ “ salt mixture.

100 per cent by weight. 100 per cent by weight.

“Accessories.”

4. Liver extract (= 0.4 gm. of dried liver).

5. Yeast 200 mg. 6. Cod liver oil concentrate 20 mg.

4. Liver extract (= 0.4 gm. of dried liver).

5. Yeast concentrate 100 mg. 6. Cod liver oil concentrate 26 mg.,

or one of the following re- placements:

(a) Cod liver oil concentrate 20 mg. + peanut oil 20 mg.

(b) Cod liver oil 20 mg. (c) “ “ “ 20 “ + pea-

nut oil 20 mg. 7. Irradiated ergosterol 0.016 mg.

1. Casein was chosen as the protein because after it is sub- jected to a process of purification, involving solution, reprecipita- tion, and washing with alcohol and ether a number of times, the fat content may be reduced below 0.2 per cent as determined by the modified Roese-Gottlieb method (Shaw, 1920). The com- mercial casein gave a value of 1.7 per cent of fatty materials. Inasmuch as good, though not optimal growth has resulted when

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McAmis, Anderson, and Mendel

casein is incorporated in certain diets to the extent of 18 per cent (Osborne and Mendel, 1926), it seemed best for the purposes of this experiment to feed the casein at a level no higher than 18 per cent, in order to keep the possible contaminants at a minimum.

2. Sucrose replaced starch, -the usual source of carbohydrate in rat diets, because starch contains not only associated fat but also combined fat which can be extracted with ether only after hydroly- sis (Taylor and Nelson, 1920). That rats would eat a diet con- sisting of a high percentage of sugar was indicated in a study by Evans and Burr (1926-27). The highest grade of granulated sugar, ground to a powder, was incorporated in the diets.

3. The salt mixture used was that of Osborne and Mendel(l919). 4. A hot water extract of pig liver,2 concentrated in a partial

vacuum at a low temperature, was given in view of the excellent growth promoted through the use of this dietary supplement, by Osborne and Mendel (personal communication). In the partially purified diet, this extract was administered incorporated with starch in small pills, while in the “fat-free” diet the extract, pre- served with alcohol, was fed as such. The amount of fatty materials in a daily dose of this extract was approximately 1.5 mg.3

5. Harris yeast concentrate, because of its lower fat content (less than 0.4 per cent of ether-soluble substances as determined by a Soxhlet extraction), replaced in the “fat-free” diet the yeast used in the partially purified diet. This lot of concentrate fed at the level of 70 mg. has been shown in this laboratory to promote the growth of rats to 120 gm. in 120 days, when it is fed as a source of vitamin B to animals depleted of their store of this dietary essential. Realizing that failure in growth might be due to the inadequacy of the yeast preparation in the so called vitamin B complex, after doubling in two cases the dose of the concentrate, we supplemented it later with 400 mg. of yeast daily.

6. The cod liver oil concentrate,4 a non-saponifiable fraction of

2 A large part of the extract used was kindly supplied by Dr. A. J. Wakeman of the Connecticut Agricultural Experiment Station.

3 This analysis was carried out by extracting in a Soxhlet apparatus with ether, strips of paper (Carl Schleicher and Schiill No. 571) on which was absorbed and dried a measured quantity of the liver extract.

4 We wish to thank Dr. H. E. Dubin of the H. A. Metz Laboratories for this concentrate, oscodal, a semisolid material from which the market

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250 Growth on “Fat-Free” Diets

that oil, kept in an amber bottle and in an atmosphere of carbon dioxide, was weighed out daily to minimize oxidation. An ex- amination of this material showed that 90 per cent of it could be recovered in the form of unsaponifiable matter.5 20 mg. of the concentrate represent approximately the vitamin potency of 2 cc. of cod liver oil (“New and Nonofficial Remedies,” 1927, 121).

7. To supply additional vitamin D, irradiated ergosterol6 was fed in preference to irradiating the animals directly, because of the great time factor involved in the daily irradiation of a large number of animals. This preparation was administered dissolved in absolute alcohol saturated with carbon dioxide, and the solu- tion, made up weekly, was kept under carbon dioxide. In deter- mining the potency of this sample of ergosterol irradiated dry, a positive line test resulted when the material was fed dissolved in olive oil in the amount of 0.001 mg. for 5 days to rats made rachitic on the Steenbock Diet 2965.7 Because, in the testing of this preparation, the irradiated ergosterol was fed dissolved in oil, it seemed advisable, in the absence of a natural fat vehicle, to feed daily a larger quantity than the test indicated potent.

A careful record of the food consumed was made, the” basal” diet being moistened to the consistency of a paste, to prevent spilling. For the duration of the greater part of the experimental period the “basal” food was weighed out daily-the food cups being filled with a gm. or two more than the individual rats had eaten the day previously, as determined by weighing the uncon- sumed food. The ‘Laccessories,” mixed all together on a small dish, were administered daily before the remainder of the food, and were usually consumed immediately-thereby lessening the possibility of deterioration of the easily oxidizable supplements.

product Oscodal Tablets is made; also for a supply of cod liver oil of the same lot used in the preparation of the non-saponifiable fraction.

5 This figure was obtained by using the modified Kerr-Sorber method

(J. Oil and Fat Ind., 3, 64 (1926)). After removal of the unsaponifiable material, the soap solution, upon acidification and extraction with ether, yielded 6 per cent of ether-soluble material, deeply pigmented.

6 This supply of tested irradiated ergosterol was kindly furnished by

Dr. C. N. Frey of the Fleischmann Laboratories. 7 For the composition of the Steenbock Diet 2965 see Steenbock, H.,

and Black, A., J. Biol. Chem., 64, 274 (1925).

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McAmis, Anderson, and Mendel 251

It is estimated from the analyses of the dietary components that those rats receiving the “fat-free” diet with the cod liver oil concentrate, received daily approximately 7 mg. of fatty ma- terials (exclusive of the 90 per cent non-saponifiable matter from the cod liver oil concentrate).

Distilled water was supplied ad Zibitum to all rats. Plan of Experiments.-The forty-seven animals used in this

study were divided into three groups-the first of which (Rats 1 to 7 inclusive) served merely as an orientation experiment. No attempt was made to secure extreme refinement of the ration fed them because this preliminary experiment was designed primarily to find out whether a cod liver oil concentrate could be utilized as a source of vitamin A when it was fed with a diet of low fat content. Interest was added to this question when Munsell and Black (1928) reported that certain cod liver oil preparations contain negligible amounts of vitamin A. Attempts were made at intervals (as indicated in Chart 1) to accelerate growth by doubling various components of the diet-yeast, liver extract, and finally the percentage of casein.

The second group of animals (Rats 8 to 21) was fed the “fat- free” diet with the cod liver oil concentrate. After about the 130th day of age, these rats received irradiated ergosterol.

The third series of animals (Rats 22 to 47 inclusive) was se- lected to answer three questions.

1. Would the growth of animals fed a supplement of cod liver oil concentrate be exceeded in rate of gain by those rats receiving the concentrate administered with a small amount of fat such as a vitamin A-free peanut oil? Hart, Steenbock, and coworkers (1926-27), and Daniels and Brooks (1926-27), have indicated that possibly the non-saponifiable fraction of cod liver oil is better utilized by an organism when the preparation is fed dissolved in an oil, than when it is administered without a fat vehicle.

2. Would better growth ensue in the rats fed cod liver oil of the same lot used in the preparation of the concentrate, than in those animals receiving the concentrate alone-both supple- ments fed at the same level, i.e. 20 mg.? The answer might give some indication of the relative potency of the two vitamin A supplements or of the deficiency of the diet.

3. Would the growth curves of animals fed a supplement of cod

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252 Growth on “Fat-Free” Diets

liver oil compare favorably with those of animals receiving the cod liver oil plus a small amount of vitamin A-free peanut oil? On the assumption that the peanut oil contains at most negligible quantities of vitamin A, any difference noted here may be said to be due to the effect of the inclusion of a small amount of fat per se or to the possible presence of other vitamins in the peanut oil.

At designated intervals, the rats were killed by illuminating gas and a necropsy performed with observations on the eyes, tongue, bones, stomach, lungs, liver, bladder, and kidneys.

Experimental Results.

I. That the particular cod liver oil concentrate used in this study, when fed at a level of 20 mg. daily, served as an effective source of vitamin A is indicated by the growth curves. Chart 1 which represents graphically the gains made by those animals fed the partially purified diet, shows that four rats (Nos. 2 to 5), receiving the cod liver oil concentrate, attained “fairly’, normal weights, while the control (Rat 7), deprived of a supplementary source of vitamin A, died, subsequent to the development of characteristic deficiency symptoms (xerophthalmia and loss in weight). One rat (No. 6) depleted of its store of vitamin A and then fed 100 mg. of the concentrate daily during 7 days, followed thereafter by 20 mg. daily, showed a clearing up of the eye dis- order and a resumption in growth. Rat 1 which received 20 mg. of the concentrate daily during the first 7 days of the experiment only, has a curve of “average” growth. This record might be taken as an argument for the storage of the concentrate, inasmuch as the other sources of vitamin A available, namely the impurities of the diet and reserve store from the mother, did not suffice for continued growth in Rat 7 which received the same diet, without the initial administration of the concentrate.

Of the animals on the purified ration, two control animals of Group II, deprived of a source of vitamin A died, showing the characteristic symptoms of vitamin A deficiency; whereas two other rats, depleted of their store of vitamin A and then receiving the concentrate, made good recovery (Chart 2). Four animals (Rats 28,45, 24,44) also resumed their growth when the cod liver oil preparation was administered after vitamin Adepletion. Rat 27, receiving daily 100 mg. of the non-saponifiable fraction of cod

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254 Growth on “Fat-Free” Diets

liver oil during the 2 days in the beginning of the experiment, and Rat 43, 20 mg. daily during the first 7 days of the experimental period, both showed delayed onset of the symptoms of deficiency due to vitamin A, as compared with their litter mates, Rats 28 and 45 respectively, similarly grown with the exception of the initial feeding of a small amount of the cod liver oil concentrate (Chart 3).

i f’i ‘\ i I I I

GROWTH ON “FAT FREE” DIET

-REPRESENTS GROWTII ON “FAT Fwe” DIETWI CONCENTRATE OF COD LIVER OIL

“*REPRESENTS &STING PERIOD

CHART 2.

From these data, it seems reasonable to assume that the non- saponifiable fraction of cod liver oil, fed at a level of 20 mg. daily, was an effective source of vitamin A. The possibility that the effectiveness of the concentrate is due to some contamination by the occlusion of a small amount of cod liver oil, is not entirely ruled out. If it is assumed that the 10 per cent of the cod liver oil concentrate not recovered in the unsaponifiable fraction (in our determination of the unsaponifiable matter in the concentrate) was all cod liver oil, this would mean that a rat might possibly

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McAmis, Anderson, and Mendel 255

receive 2 mg. of cod liver oil in the daily ingestion of 20 mg. of concentrate. If the growth of the rats were limited by the cod liver oil supplement alone, then one should expect much better growth in those animals receiving 20 mg. of cod liver oil-10 times the amount possible in the case of those rats fed the con- centrate. From Charts 4 to 6, it is seen that there is only a very little difference in the growth curves of animals fed supplements of cod liver oil and the concentrate of that oil respectively.

ROUPI

****REPRESENTS GROWTH ON “FAT FREE’ DW -REPRESENTS GRWW ON “FAT Frtce” DIET WITH

CONCENTRATE OF Coo LIVER OIL

=.-REPRESENTS FASTING PERIOD XTNDICATES TNITIAL EYE SYMPTOM~J

CHART 3.

II. The effect of an addition of a small amount of fat is illus- trated from a study of the growth curves in Charts 5 to 8. From a comparison of Charts 5 and 6, it is seen that the animals receiv- ing cod liver oil demonstrated growth a little better than those fed the same weight of the concentrate made from this lot of cod liver oil. Slightly better growth was made by those rats receiv- ing a drop (about 20 mg.) of peanut oil in addition to the concen- trate (Chart 7) than those given the concentrate alone (Chart 5)

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Growth on “Fat-Free” Diets

-

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l-l I I- REPRESENTS GROWTN ON “FAT FREP~ DIET WITH CONCENTRATE OF COD LIVER OIL

1.11 REPRESENTS I%STINP RRIOD

+ &INDICATES INITIATION OF ERGOSTEROL

Y.C. INDICATES DOUBLING OF YEAST CONCENTRATE

CHART 4.

I I I GROUPIi

3f-i I-&PuESeNTs GROWTH ON “FAT FREE”DIET WITW

i CONCENTRATE OF Coo LIVER OIL

d . -~REPResENTS FASTING RRIOD *n

CHART 5.

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McAmis, Anderson, and Mendel 257

-REPRESENTS GROWTH ON WF~~F~~~"D~~~ WITH

&D LIVER OIL m-

..‘ REPRESENTS FASTING RRIOD

CHART 6.

-RLPRESCNTS GROWTH ON “FAT FRCE*'DIET WITH

CONCENTRATE OF Coo LIVER OIL AND PEANUTOIL -- --REPRESENTS GROWTH ON “FAT FREE"DIET WITH

CHARTS.

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258 Growth on “Fat-Free” Diets

and comparable growth was made by the former rats and those animals receiving only the cod liver oil. However, the best growth curves obtained represent the gains made by those rats whose diet was supplement.ed by 1 drop each of cod liver oil and peanut oil. Whether the beneficial effect of the addition of peanut oil is due to a small amount of vitamins (known or un-

GROWTH ON “FAT FREE”

COD LIVER Chx~ AND PEANUT --- -

--REPRESENTS GROWTH ON “FAT FREE”

PEANUT OIL ALONE -- ~==REPRESENTS FASTING PERIOD

DIET

OIL -

DIET

CHARTS.

known) contained in it; to the aid fat may render in the absorption of the fat-soluble vitamins; or to the presence of the fat per se, is undetermined. The vitamin A content of the peanut oil was not enough to prevent decline in weight of the three rats adminis- tered 1 drop of this peanut oil as a sole source of vitamin A, and the animals did resume growth when the oil was supplemented with a vitamin A-bearing material (see Charts 7 and 8). Peanut

oil incorporated in the diet to a very much larger extent-22 per

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McAmis, Anderson, and Mendel 259

cent-seems to indicate that this oil does not contain any appreci- able amount of vitamin A (Holmes, 1923).

A comparison of the growth curves of animals fed supplements of cod liver oil concentrate, alone, and with peanut oil, and cod liver oil both with and without peanut oil, indicates that those animals receiving the most fat grew best.

III. A few outstanding fiddings were noted in the necropsies: 1. A gelatinous mass was found in the bladders of one-half of

the animals grown with the cod liver oil concent,rate as compared with only one out of five of the animals receiving supplements of either cod liver oil or cod liver oil plus peanut oil. Some of these masses were very soft, while others were rather firm and resembled stones.

The kidneys of half the animals on the “fat-free” diet plus the cod liver oil concentrate were mottled, and in some cases, there were indentations in the surface of the kidney. Urinary calculi were observed in three rats (Nos. 6, 8, 19) first depleted of their store of vitamin A and then fed cod liver oil concentrate. The stones were found, in two of the animals, in the pelvis of the kid- ney, and in the third rat, several large stones were in the bladder and one small stone in the right ureter. The right kidney of this animal was only a shell, after the removal of the green pus which filled it (Fig. 1). These findings suggest that all damage due to dietary deficiencies is not completely repaired upon supplying the lacking component of the food-an illustration of a fact not always appreciated in the quantitative tests of the vitamin content of foods, as all “curative” methods may be rendered ineffective by previous irremedial conditions due to the depletion in an organism of a dietary essential.

2. Abnormality in the appearance of the lungs was observed in more than half of the rats receiving from the beginning of the experimental period the cod liver oil concentrate without addi- tional fat, whereas none of the other animals administered a source of vitamin A with fat showed gross changes in this organ.

3. Hair balls were present in the stomachs of eleven rats out of the forty-seven observed. Mitchell, Bradshaw, and Carlson (1924) state that ‘(formation of hair balls is attributed to high fat, content and sticky consistency of the food.” Our observa- tions can at most confirm only the latter port.ion of their state-

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260 Growth on “Fat-Free” Diets

men&-namely, that referring to t.he sticky consistency of the food.

On the living animals, grown on the <‘fat-free” diet with the cod liver oil concentrate, certain observations were made-such

I, It

FIG. 1. Kidneys from rat (So. 6) grown on the partially purified dieb and fed cod liver oil concentrate, onl?J c~Jler depletion of the body’s store oJ vita-

min A.

FIG. 2. Eye conditions observed. A, Rat 28, “control,” grown on “fat- free” diet-just before administration of the cod liver oil concentrate. Age 59 days; weight 106 gm. B, Rat 14, grown on the “fat-free” diet with cod liver oil concentrate. Age 132 days; weight 125 gm. Xote the rim about the edge of the eyelids. C, Rat 0, “normal,” grown on a diet in which 40 per cent of the calories was furnished by dried skimmed milk and 60 per cent by soy bean oil. This diet was supplemented by 400 mg. of yeast and 1 drop of cod liver oil. Age 58 days; weight 125 gm.

as poor condition of the fur, and in some cases priapism and bloody urine. In one rat,, about 4 inch of the end of the tail was quite dark and appeared gangrenous. A peculiar condition about the eyes resulted in three of the rats fed the purified ration containing the non-saponifiable fraction of cod liver oil. The eyelids were pink and in general the eyes resembled those in the incipient

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McAmis, Anderson, and Mendel

stages of xerophthalmia, but at no time was a picture of true xerophthalmia presented (Fig. 2).

Inasmuch as most of the abnormalities noted occurred in those animals receiving the least fat, the argument for the beneficial effect of fat is strengthened.

IV. The fact that, regardless of the supplements used, optimal growth did not result in the animals reared on a diet extremely poor in fats, might be attributed to the inadequacy of one or more components of the diet. The inclusion of a source of vitamin E might have promoted growth (Evans, 1928). Likewise the feed- ing of casein at a higher level might have made possible an increase in body weight. The limit of the ability of the rat to utilize as high a concentration of sucrose as this diet contains may have been overstepped, thereby rendering less efficient some of the ingested food. Those animals in Group III fed the cod liver oil prepara- tion do not show better growth than the rats in Group II in spite of the fact that in the former group irradiated ergosterol was administered throughout the experimental period, whereas the other rats received it only after about the 130th day (see Charts 4 and 5). Possibly irradiated ergosterol may not be utilized when it is fed in a non-fat medium as in a diet devoid of fat. The ani- mals, however, did not appear to be rachitic, as was to be expected inasmuch as a good salt mixture was used. Irradiation of the animals directly probably would have been more satisfactory but even then the ability of an organism to respond to the irradia- tion to which it is subjected might be lessened when the animal is grown on a diet extremely poor in fatty materials. The inade- quacy of the source of the vitamin B complex might account for the growth of the animals on the purified diet being inferior to that of the rats fed the partially purified diet. However, the addition of 400 mg. of yeast, from the same lot fed to the rats which were receiving the partially purified diet, did not produce marked increase in growth (Chart 4, Rats 13 and 20). Unfor- tunately, it is not known what type of growth curve would have resulted had the yeast been fed as a source of vitamin B compo- nents from the beginning.

SUMMARY AND CONCLUSION.

In order to study the composition of body fat yielded by an animal grown under a dietary regime in which there was no in-

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262 Growth on “Fat-Free” Diets

gested fat, a diet of extremely low fat content (consisting of ex- traoted casein, sucrose, Osborne and Mendel salt mixture, yeast concentrate, cod liver oil concentrate, irradiated ergosterol, and hot water extract of liver), has been fed to albino rats. Compara- tively good, though by no means optimal growth has been recorded for these animals. The best growth, however, was exemplified by those animals which received some small inclusion of fat in their diet. Whether this apparent beneficial effect of a small amount of fat is due to its content of vitamin A or other vitamins, or to its action as a vehicle for the fat-soluble vitamins, or whether fat per se is essential, is not conclusively demonstrated.

BIBLIOGRAPHY.

Daniels, A. L., and Brooks, L. M., Proc. Sot. Exp. Biol. and Med., 24, 972 (1926-27).

Evans, H. M., J. Nutr., 1, 23 (1928). Evans, H. M., and Burr, G. O., Proc. Sot. Exp. Biol. and Med., 24, 740

(1926-27). Hart, E. B., Steenbock, H., Kletzien, S. W., and Scott, H., J. Biol. Chem.,

71, 271 (1926-27). Holmes, A. D., J. Metabol. Research, 3, 583 (1923). Mitchell, H. S., Bradshaw, P. J., and Carlson, E. R., Am. J. Physiol., 68,

203 (1924). Munsell, H. E., and Black, H., J. Am. Pharm. Assn., 17, 139 (1928). Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 37,557 (p. 572) (1919). Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 69,661 (1926). Shaw, R. H., J. Ind. and Eng. Chem., la,1168 (1920). Taylor, T. C., and Nelson, JI M., J. Am. Chem. Sot., 42, 1726 (1920).

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and Lafayette B. MendelAva Josephine McAmis, William E. Anderson

DIETSGROWTH OF RATS ON "FAT-FREE"

1929, 82:247-262.J. Biol. Chem. 

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