THE INFLUENCE OF BUTTER-FAT ON GROWTH.’

BY THOMAS B. OSBORNE AND LAFAYETTE B. MENDEL,

With the Cooperation of EDNA L. FERRY and ALFRED J. WAKEMAN.

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

in Yale University, New Haven, Connecticut.)

(Received for publication, November 4, 1913.)

We have recently pointed out2 that while young rats grow for a time at a normal rate on the “protein-free milk” diet used in our earlier experiments, they sooner or later cease to grow, so that they rarely attain more than two-thirds of the weight normal for fully grown rats receiving a diet chiefly composed of milk and lard. Furthermore, we showed that rats which had ceased to grow and were declining on a “protein-free milk” diet, at once recovered and resumed a normal rate of growth when a part of the lard in their food was replaced by a quantity of unsalted butter corresponding to that in the milk-food. The striking way in which butter, thus supplied, influenced the growth of these young rats made it evident that it furnishes some substance which exerts a marked influence on growth.

These observations have since been verified by numerous addi- tional experiments and an attempt has been made to determine with which of the components of the butter this growth-promoting power is associated. As is well known, butter consists of about 82-83 per cent of the glycerides of numerous fatty acids, about 15 per cent of water containing each of the soluble constituents of milk, and from 1 to 2 per cent of solid matter, consisting chiefly of cellular debris from the mammary glands, bacteria, calcium

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

* Osborne and Mendel: The Relation of Growth to the Chemical Con- stituents of the Diet, this Journal, xv, pp. 311-326, 1913.

423

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424 Influence of Butter-fat on Growth

phosphate, particles of casein, and accidental impurities intro- duced during the process of making the butter.

In view of the possibility that even an extremely minute quan- tity of some substance might exert the favorable influence on growth observed in all of our experiments, we separated the butter into three parts, nameIy the fatty substances, the insohrble sohd elements, and the aqueous solution containing lactose, soluble inorganic salts and other soluble components of the milk; and by feeding trials found that the growth-promoting factor was contained in the fat fraction. Further consideration of the two other fractions is therefore unnecessary.

The butter-fat used in the analytical, as well as the feeding experiments here described, was prepared as follows. The butter was melted by heating in a flask immersed in a bath of water not exceeding 45”, and centrifugated for about an hour at a high speed. The melted butter was thus separated into a layer of perfectly clear fat, an opalescent aqueous layer, and a deposit of white solid matter. The clear fat was sucked off with care, and thus separated from all of the other parts of the butter. By this method the use of all solvents was avoided, and any substances which might have been dissolved thereby from the other parts of the butter were excluded.

So much has been written about the significance of phosphatides (lecithin, etc.) in various biological phenomena, and in growth among others, that a careful analysis of large quantities of the butter-fat was instituted to detect the presence of members of this group of so-called lipoids. The butter-fat prepared as described was found to be entirely free from nitrogen and phosphorus and was devoid of any ash-yielding, or water-soluble, components. The absence of phosphatides from the product corresponds with the recent statement of Njegovan3 who con- cludes that milk contains no lecithin whatever, and suggests that the contradictory claims of other investigators are attributable to inadequate methods of analysis.

Butter-fat, thus prepared, has proved to be quite as effective as butter or milk in promoting the recovery and renewed growth of animals which have ceased, or failed, to grow on the natural

3 Sjrgovan: Biochem. Zeitschr., [iv, p. 78, 1913.

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T. B. Osborne and L. B. Mendel 425

“protein-free milk” dietaries in which lard furnished the fat component. Our previously published experiments4 in which sim- ilar recoveries were made when butter replaced a part of the lard of diets containing the “artificial protein-free milk” are compli- cated by the fact that butter contains about 15 per cent of butter- milk, and hence the improvement shown by such experiments might be attributed to some constituent of the buttermilk. Ex- periments with “artificial protein-free milk” and butter-fat are in progress and it is hoped by these to learn whether or not accessory substances other than those contained in the butter- fat are necessary for growth. These experiments are not yet completed.

Charts showing the body-weights of growing rats that had begun to decline on our “protein-free milk” food mixtures, and were supplied with foods having butter or butter-fat introduced,5 are appended (see Charts I, II, and III). The composition of the mixtures was as follows:6

Butter foods Butter-fat foods per cent per cent

Protein.................................... 18 18 Starch..................................... 36 26 Protein-free milk., 38 28 Lard...................................... 19 10 Butter-fat................................. 15.3\ Buttermilk, etc.. . . . . . . . . . . 2.i!

butter ‘:

The efficiency of the butter-fat, or some component thereof, in specifically promoting growth is further shown in another way. As has already been pointed out, when very young rats are placed on a mixture of purified protein, lard, starch, and “protein-free milk,” prepared in imitation of the gross composition of the highly successful milk-food, they show a varying capacity to grow. In some cases growth has st.opped after sixty days; other animals have continued to grow for one hundred days or more. But in the growth experiments there has always been an inevitable ulti-

3 This Journal, xv, p. 326, 1913. 5 For similar recoveries induced by milk-food, we Charts II and III,

this Journal, xv, pp. 321-322, 1913. 6 For details regarding the milk-foods used in our experiments. and other

comparisons, see Osborne and Mendcl : this Jownal, xv, p. 318, 1913.

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426 Influence of Butter-fat on Growth

mat.e inhibition of growth, and nutritive decline, connected with some diet, factor. Very few of our rats grew after they were 140 days old, at which age two-thirds of the normal growth of the male, and three-fourths of that of the female is usuaIly made. We have accordingly attempt,ed to learn whether the continued exhibition of butter-fat from very early in the growth period would enable the animals to attain their normal maximum weight and thereby avert the invariable failure which hitherto was met with sooner or later. If it be assumed that in these earlier experiences the substance, or substances, essential for growth, and supplied inadequately, or not at all, in the artificially prepared diets, is furnished by a reserve stored in the cells of the young animals, this must be exhausted after a time, and lead to failure of growth. If, however, a growth-promoting substance is present in the butter- fat, and the latter is supplied in abundance, growth ought to continue to its logical conclusion, in the absence of other inhibi- tory factors.

Experiments to test this were begun with young rats whose diet from an early period consisted of food of the following com- position :

per cent Purified protein.............................................. 18 Starch., . 26 Protein-freemilk............................................ 28 Lard.. 10 Butter-fat................................................... 18

In harmony with the theory outlined above, the outcome of these newer feeding trials already demonstrates a far more success- ful continuance of growth on the foods containing butter-fat than on any other artificial diet-mixture (except milk-food) hitherto tested. An illustrative chart (IV) is appended.

In further corroboration of the efficiency of the butter-fat, in promoting growth, and part,icularly after a previous decline, we may cite additional experiments involving the use of centrifugated, or so-called skimmed milk as the basis of the ration. A food paste, intended to resemble our successful milk-food, was pre- pared by the use of dried centrifugated milk.’ The product used

?Like the whole milk this was supplied to IIS in poxvdered form by the Merrell-Soule Company of Syracuse ;N. Y.

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T. B. Osborne and L. B. Mendel 427

contains only 1.18 per cent of fats. The food had the following composition :

per cent Dried centrifugated milk. _. . . _. . . . . . . . . . . . 44 Starch....................................................... 28 Lard.. . . . . . 28

This food, although not entirely free from milk-fat, contained only 0.52 per cent, i.e., less than one twenty-fifth as much as the whole milk-food used for our earlier experiments. Although rats have grown normally upon our whole milk-food for more than a year, the centrifugated milk-food has in every case failed to prove adequate for a comparable period. It is true that the centrifugated milk-food has been more satisfactory as a growth ration than our “protein-free milk” foods, i.e., the moment of ultimate failure to grow or to decline has been postponed longer. This may well be due to the small quantities of the essential butter-fat still present in the commercial product used by us. However, the substitution of a part of the lard in the food-pastes by an amount of butter equivalent to that naturally present in our milk-foods brought prompt recovery, as exemplified in the appended chart V.

The outcome of these experiments clearly indicates that the growth-promoting substance of the milk is to be found in the butter-fat fraction thereof; for the two rations here illustrated and characterized by either nutritive failure or success, differ in respect to the fat component only. The influence of heating and other processes involved in the preparation of milk for food were also incidentally investigated. These studies, though far from com- pleted, have given no evidence, in so far as nutrient efficiency is concerned, of a damage to the centrifugated milk by vigor- ous sterilization. We hope to return to this question in a later communication.

The experiments recently reported by McCollum and Davis,* who added an ether extract of butter or eggs to artificially pre- pared food-mixtures, are in accord with the results which we have obtained by feeding butter, or butter-fat, to rats which were declining after a period of growth on a diet of isolated food-sub-

* Cf. McCollum and Davis: The Necessity of Certain Lipins in the Diet during Growth, this Jozunal, xv, p. 167, 1913.

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428 Influence of Butter-fat on Growth

stances. In none of their published records was the recovery so rapid as in most of ours, nor was the rate, or extent, of growth, after reaching the previous maximum weight, any greater than on the butter-fat-free diet earlier supplied. In the experiments they report, the gain made by their rats after recovering to their previous maximum weight was 40, 25, 40, and 63 grams, a part of the latter gain being made after the rat had eaten her own young. The time during which the rats were fed on diets con- taining the ether extract was 5, 9, 12, and 12 weeks, but in no case did the animal reach a weight normal for its age. Although the data furnished by McCollum and Davis strongly indicate that butter-fat has a marked influence on growth they by no means prove that butter-fat contains something essential for the metabolism of growth, apart from that of maintenance. The added butter-fat may have simply supplied something analogous to the so-called vitamines, which Funk considers to be essential for life, and thereby enabled the animals to resume growth on a food thus made adequate for maintenance. Until it is shown that rats can be maintained for long periods on such diets as McCollum and Davis used no final conclusion can be drawn respecting the above question.

By numerous experiments we have shown that mature rats can be maintained on our “protein-free milk” diets for more than a year, and that young rats on similar diets containing proteins inadequate for growth can be maintained nearly as long. Such foods consequently supply all that is essential for maintenance

alone. Since growth ceases on these foods after a comparatively short time, and is at once resumed and continued throughout the entire period of normal growth when a part of the lard is replaced by butter-fat, it is almost certain that butter-fat contains some- thing essential for growth in addition to what may be required for maintenance. This recovery and renewed growth must be attributed to somct,hing which distinguishes butter from the ordi- nary fats, for not only do lard and olive oil9 lack this growth- promoting power, but young rats grow on our “protein-free milk” foods when all of the lard is replaced by carbohydratelO and no ether-soluble substances are present in the food.

“McCollum and Davis: lot. cit. lo Cf. Osborne and IIendcl: Feeding Experiments with Fat-free Food

Rlixturcr, this Journal, xii, pp. 81-89, 191%.

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T. B. Osborne and L. B. Mendel 429

It thus appears improbable that glycerides of the fatty acids ordinarily present in foods are responsible for the promotion of the growth observed when butter-fat replaces lard in the diet of rats which have ceased to grow. Lecithin and other phosphorus- or nitrogen-containing substances are excluded by the absence of phosphorus and nitrogen from our butter-fat; and cholesterol by the fact that even more of this substance has been obtained from lard than from butter.”

So far as our experience has shown, the addition of butter-fat to our natural “protein-free milk” foods gives them an efficiency quite comparable with that of our milk-food in promoting recovery and the completion of growth. The exact chemical differences between the adequate butter-fat and the inadequate lard (which determine success and failure respectively in the food-mixtures employed) are far from being satisfactorily known. Chemical examination of the butter-fat indicates that the effective compo- nent is not a phosphatide or any inorganic substance, inasmuch as nitrogen, phosphorus and ash are lacking in the product em- ployed. It is suggestive to note that in the one case (lard) we are dealing essentially with a fat-mixture deposit,ed in storage depots of the animal organism; in the other, t.he butter-fat repre- sents the product of met’abolic activity and synthesis on the part of the cells of the mammary gland. What, if anything, this dis- tinction between cellular product and reserve fat may mean physiologically, remains to be investigated.

The researches which have been devoted in recent years to certain diseases, notably beri-beri, have made it more than prob- able that there are conditions of nutrition during which certain essential, but, as yet, unknown substances must be supplied in the diet if nutritive disaster is to be avoided. These substances apparently do not belong to the category of the ordinary nutrients, and do not fulfil their physiological mission because of the energy which they supply. Funk has proposed the name vitamine for the type of subst’ance t.hus represented.‘?

Without minimizing the importance of the new field of research and the new viewpoints in nutrition which are present’ed by these

I1 Cf. McCollum and Davis: Zoc. cit., p. li4, who fed cholesterol to rats. I2 The literature on the subject has been reviewed by Funk: Ueber die

physiologische Bcdcutung gcwisser bisher unbekannter Sahrungsbestand- teile der Vitamine, EryeB. d. Physiol., xiii, p. 125, 1913.

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430 Influence of Butter-fat on Growth

recent findings, we may nevertheless hesitate to accept the extreme generalizations which have already been proposed on the basis of the evidence obtained largely from the investigation of patho- logical conditions. The statement, for example, that a “ tadellose Nahrung” may prove entirely inadequate unless “vitamines” are present, at once suggests a series of questions bearing on what is included in the new term. It is still rather early to generalize on the r81e of accessory “vitamines” when the ideal conditions in respect to the familiar fundamental nutrients and inorganic salts adequate for prolonged maintenance are not completely solved. Speculation is quite justifiable in so far as it directs attention to a new phase that needs to be taken into account.

Funk has expressed the belief that the substance which pro- motes growth and must be present in order to avert the cessation of growth, which we have described to occur after a certain period of successful growth on our earlier dietaries, is either identical with, or analogous to, the “vitamine” which plays the r81e of an ant’iscorbutic substance. For this we can as yet find no compelling evidence. Certainly the nitrogen-free butter-fat, so successful in remedying our growth failures, contains no substance chemically related to the nitrogenous products which have lately been cred- ited wit’h this unique physiological e&iency.13 Furthermore it

la Cf. Funk: Ueber die physiologische Bedeutung gewisser bisher unbe- kannter Nahrungsbestandteile der Vitamine, Ergeb. d. Physiol., xiii, p. 130 et seq., 1913. In reviewing our earlier published experiments Funk has erroneously assumed that we secured completed growth with the diets in which the butter component was not yet employed. It is true that the increments in weight were in some cases very noteworthy; but inevery instance cessation ultimately ensued before the completion of the normal progress of the growth or subsequent maintenance. We have never denied the necessity of a growth-promoting food accessory in accord with the claim of Hopkins; and recently we pointed out that the successful partial completion of growth, such as has been obtained in our experiments, may well have been due to a store of the essential compound in the body of the experimental animals at the beginning of the trials. It is by no means necessary to assume with Funk that small quantities of these accessory substances were inadvertently left in our food preparations owing to insuf- ficient extraction with alcohol.

Furthermore we cannot agree with Funk that the rat is not well adapted to experiments on the physiology of growth. The superiority of this ani- mal has been pointed out by us elsewhere (cf. this Journal, xiii, p. 233,

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T. B. Osborne and L. B. Mendel 431

is well to bear in mind that it is not improbable that the anti- neuritic and antiscorbutic constituents of foods are not identical with the substances alleged to assist in maintaining body-weight.14 FunkI has lately asserted that the simultaneous administration of at least two substances is necessary to produce the curative effect obtained in his previous experiments with the “vitamine” fraction from rice-bran or yeast. Voegtlin and Towles16 have noted that extracts of autolyzed spinal cord may be antineuritic, yet be unable to reestablish normal metabolism, i.e., restore body- weight.

Butter-fat has shown a further interesting nutritive superior- ity over lard. At certain periods of the year, particularly in summer months, we have frequently failed to secure satisfactory growth on the dietaries which proved adequate during the usual period of sixty to one hundred days at other seasons. Occasionally young rats in the stock colony have exhibited a similar “epidemic” of poor growth at the same season. The failures are, however, not common to rats fed on the milk-food; and we have lately observed that the seasonal failure is also averted by the addition of butter-fat to the usual “protein-free milk” food-mixtures.17 Again, another type of nutritive deficiency exemplified in a form of infectious eye disease prevalent in animals inappropriately fedI is speedily alleviated by the introduction of butter-fat into the experimental rations.

The chemical character of the unique LLaccessory substance” in butter-fat must be investigated in detail and its possible prcs-

1912) and is also apparently recognized by both Donaldson and MeCollum and their coworkers. We have found rats to be responsive to changes in diet; and we count it no disadvantage that the experiments must be con- tinued over sufficient time to exclude minor incidental fluctuations.

l4 Cf. Cooper: Journ. of Hygiene, xii, p. 433, 1912. 15 Funk: Ergcb. d. Physiol., xiii, p. 547, 1913; &it. Med. Journ., April

19, 1913; Jown. of Physiol., xlvi, p. 173, 1913. I6 Voegtlin and Towles: Journ. oj Exp. Pharmacol., v, p. 67, 1913. 1’ These summer failures in growth have been reported to us by

colleagues to occur likewise in other institutes. 18 Cf Knapp: Experimenteller Beitrag zur Ermihrung von Ratten mit

kiinstlicher Nahrung and sum Zusammenhang von Ernahrungsstdrungcn mit Erkrankungen der Conjunctiva, Zeitschr. f. ezp. Pnth., v, pp. 147-170. 190s.

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432 Influence of Butter-fat on Growth

ence elsewhere determined. Experiments are already under way with varying proportions of butter-fat in the ration; but we have not thus far determined the necessary allowance. On ’ the other hand, no amount of butter-fat will induce growth on certain die- taries in which the proportions and nature of 6he inorganic salts are inappropriate (as in our Salt mixture I),lg or the quantity and character of the protein is inadequate. The ‘lBausteine” must not be overlooked in our enthusiasm for these newer features.

ADDENDUM. An investigation now under way to determine the possible efficiency of fats other than butter-fat in preventing decline on our protein-free milk-food and promoting growth in the way that butter does, has already indicated marked differ- ences in fats from different sources. Egg yolk-fat, for example, appears to behave like butter-fat; some other oils have thus far proved no more efficient than lard. Such considerations make it evident that the comparative value of the natural fats em- ployed in nutrition must be determined, as well as the individual role of the different proteins, carbohydrates, and mineral nutrients.

10 See Osborne and Mendel: Carnegie Institution of Washington, Publi- cation 156, pt. ii, p. 80.

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T. B. Osborne and L. B. Mendel

CHART I. Curves of body-weight of rats which have ceased to grow and have declined on foods containing the natural “protein-free milk,” and have recovered when 18 per cent of unsalted butter replaced the same quantity of lard in the diet, as indicated by the interrupted lines (-0-o-o-o). The proteins furnished in the different experiments were as follows: casein, Rats 1204, 1268, 1276, 1281, 1292; ovalbumin, Rats 1268, 1276.

The ordinates represent grams of body-weight., as indicated. The divi-

sions of the abscissa represent 20-day periods.

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434 Influence of Butter-fat on Growth

CHART II. Curves of body-weight of male rats which have ceased to grow and have declined on foods containing the natural “protein-free milk,” and have recovered when 18 per cent of bulter-jut replaced the same quantity of lard in the diet, as indicated by the interrupted lines (-0-o-o-o). The proteins furnished in the different experiments were as follows: casein, Rats 1224, 1235; edestin, Rat 1391; zein + casein, Rat 1616.

The ordinates represent grams of body-weight, as indicated. The divi- sions of the abscissa represent 20-day periods.

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T. B. Osborne and L. B. Mendel 435

___--.---- 4 __--- _*

wth Naxral 'rote;,,+ + Butter-fat ?

CHART III. Curves of body-weight of female rats which have ceased to grow and have declined on foods containing the natural “protein-free milk,” and have recovered when 18 per cent of butter-jut replaced the same quantity of lard in the diet, as indicated by the interrupted lines (-0-o-o-o). The proteins furnished in the different experiments were as follows: casein, Rat 1217; zein + la&albumin, 1186.

The ordinates represent grams of body-weight: as indicated. The divi- sions of the abscissa represent 20-day periods.

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436 Influence of Butter-fat on Growth

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CHART IV. Typical curves showing prolonged normal growth of white rats on foods containing 18 per cent of butter-jut. The proteins furnished in the different experiments were as follows: casein, Rats 1592, 1599, 1619, 16.36, 1652, 1655, 1657; edestin, Rats 1650, 1651.

The ordinates represent grams of body-weight, as indicated. The divi- sions of the abscissa represent Z&day periods.

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T. B. Osborne and L. B. Mendel 437

160

-I CHART V. Curves of the body-weight of rats which have ceased to grow

and have declined on the centrijugated-milk-food, and have recovered when 18 per cent of butter-fat replaced the same quantity of lard in the diet.

The ordinates represent grams of body-weight, as indicated. The divi- sions of the abscissa represent 20-day periods.

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

Thomas B. Osborne, Lafayette B. Mendel andGROWTH

THE INFLUENCE OF BUTTER-FAT ON

1914, 16:423-437.J. Biol. Chem. 

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