Unidentified Nutrients

REMARKABLE progress has been made in identifying the nutrients in our foods and feeds, but results of work in various laboratories indicate that there are still other unidentified constituents in some foods and feeds that may play an important role in nutrition.

Altogether, the findings—with the rat, mouse, dog, monkey, and the human—constitute a body of evidence that should be regarded realistically in considering the question of whether we are well fed.

Here we shall present evidence obtained in the Bureau of Dairy Industry laboratory at Beltsville that we believe demonstrates that:
   Milk and certain other foods and feeds contain an unidentified nutrient;
   White flour, enriched white flour, whole-wheat flour, yeast, and certain other foods and feeds do not contain this nutrient;
   When the young are deprived of this factor and then are fed a diet containing all known nutrients in adequate amounts or a diet containing these nutrients along with white flour, enriched white flour, whole-wheat flour, or yeast, their growth and development is by no means normal and under certain circumstances is impossible, unless they are supplied in some way with this unidentified factor.

The nonfat solids of milk are a good source of protein, calcium, and riboflavin, and contain other salts and known water-soluble vitamins of value in nutrition. These nutrients supplement the foods used as our principal sources of energy, such as bread, butter, oleomargarine, sugar, cereals, and so on. We believe, however, that these foods can be supplemented with all of these nutrients or with all known nutrients and not produce the effect—on growth, for example—that is produced by supplementation with milk itself or with dried skim milk. What we have said here about flours appears to be true of the grains in general; they are deficient in this unidentified nutrient. With livestock, this unidentified nutrient is supplied by roughages and leafy feeds.

In one experiment, we fed rats as much as they would eat of certain rations, as follows:
   Group 1. A basal ration containing adequate amounts of all known nutrients;
   Groups 2, 3, and 4. The same basal ration, containing 45.5 percent of white flour, enriched white flour, or whole-wheat flour, respectively, in place of carbohydrate;
   Groups 5 and 6. The same enriched white flour and whole-wheat flour rations as fed to groups 3 and 4, except that the ration contained 10 percent of dried skim milk.

All of these rations contained amounts of all known nutrients that should be adequate for optimum growth. Nevertheless, the average growths of the rats in groups 1, 2, 3, and 4 were all about the same, and were not much more than half that of their sex-litter mates in groups 5 and 6, which received the dried skim milk. There is no question about the statistical significance of these results; we shall consider their interpretation and significance relative to the nutritive value of milk.

Some workers have reported that their rats do not eat a ration of enriched white flour bread as well as they do a bread made from this flour supplemented with milk solids. It is possible that our basal ration and the rations containing the white flour, enriched white flour, and whole-wheat flour were “unpalatable,” and that the addition of the dried skim milk made these rations more “palatable.” Unquestionably this was, in a sense, a fact; why then were these rations without the skim-milk powder in them unpalatable to our rats? Was this unpalatability due to some quality of these rations irrespective of the nutrients in them; or was it a response, which is familiar, of animals depleted in respect to some particular nutrient, to a ration deficient in that nutrient? If the latter is the explanation, might it not be possible to change the “palatability” of these rations to our rats, not by altering the rations themselves, but by correcting the nutritional deficiency in the test animals? That we tried to do.

In related experiments we fed 333 pairs of litter-mate male rats and 112 pairs of litter-mate female rats exactly the same basal ration as the one used with the rats previously mentioned. In these experiments one rat in each pair received in addition a few milligrams daily of a liver extract.  Here, as before, the rats on the unsupplemented basal ration grew at a rate that was not much more than half of normal. By “normal growth” is meant the growth of our rats that received the basal ration plus an amount of the liver extract adequate for optimum growth. But the feeding of the liver extract, which was given separately from the basal ration, improved the palatability of this ration, increased consumption, and nearly doubled the growth. In fact, it was not even necessary to feed the liver extract to obtain the results; we obtained exactly the same results when the liver extract was injected under the skin or into the muscles of our rats. And the same results were also obtained when, instead of a few milligrams of liver extract, a few micrograms—a few millionths of a gram—of concentrated preparations of the growth-promoting material in these liver extracts were fed daily, separate from the basal ration.

The results show also that the administration of the liver extract separate from our basal ration brought about the same growth as the feeding of dried skim milk incorporated in this ration. It is obvious that the unpalatability of our basal ration can be just as effectively overcome in either of these ways, and that it is due to a nutrient deficiency in the basal ration and in our test animals.

But what nutrient was involved, and why were our test animals deficient?

We stated that the basal ration used in the experiments contained adequate amounts of all known nutrients for normal growth. The statement covers quite a bit of territory. We have found that our basal diet and the wheat-flour diets were deficient in some nutrient that can be supplied in a liver extract or in skim-milk powder. Before concluding that this nutrient actually is some still unidentified factor, let us consider what we mean by “all known nutrients,” and how we have arrived at the conclusion that our basal rations is “adequate for normal growth” in respect to these nutrients.

There is nothing unique about the composition of what we have called “our basal ration.” It is a ration often used in the biological assay of vitamin A, except that we added vitamin A to it. The minerals in the ration were supplied by a common mineral mixture that unquestionably is adequate; the B vitamins were supplied by 10 percent of yeast. No increase in the quantity or change in the kind of yeast improved the growth of our rats on this ration, and, as a result of many efforts to supplement the ration with various amounts of crystalline or pure vitamins, or to replace the yeast by mixtures of these vitamins, we concluded that our basal ration also supplied adequate amounts of all of the available fat-soluble and water-soluble vitamins—vitamins A; D, E, K, and C (ascorbic acid) ; choline, thiamine, riboflavin, pyridoxin, pantothenic acid, nicotinic acid, para-aminobenzoic acid, inositol, biotin, folic acid (or pteroylglutamic acid), also xanthopterin.

The protein in our basal ration (25 percent) was comprised of about 5 percent of yeast protein and 20 percent of casein, the principal protein in milk. Both proteins are ordinarily considered complete and the total amount adequate, but the casein we used had been heated for long periods with successive lots of alcohol as had been our custom in removing vitamin A from it. This method of removing the vitamin A was one that was formerly used in vitamin A assay work. Could our handling of this casein have injured its nutritive properties?

This question led to a study of the effect of using, in our basal ration, casein preparations made in various ways. One preparation that was being used in vitamin-assay work in another laboratory, and that had been quite extensively washed with acidulated water and then extracted with alcohol for 24 hours, gave results similar to our casein; one commercial “vitamin free” casein gave hardly significantly’ better growth than our casein; another commercial “vitamin free” casein, and other caseins prepared by milder treatments with alcohol, or with alcohol and ether, all gave decidedly better growth than our casein.

None of these caseins gave normal growth. But samples of “commercial” caseins that were tested gave almost normal growth, and a casein precipitated from skim milk by an 11-day dialysis against water and another centrifuged out of milk at 50,000 revolutions a minute and repeatedly washed and re-centrifuged, gave fully normal growth.

These results certainly mean either one of two things: That many researches are now being conducted with caseins in which the nutritive value has been impaired by various methods of treatment—in which case our casein would be one that had been the most impaired of any of those tested, or that many researches are being conducted today with caseins containing as an impurity more or less of some unidentified nutrient—in which case our casein would be one of those containing the least amount of this nutrient.

1. Replacing our casein in our basal ration with an equal amount of heat-coagulated egg albumen (with or without a supplement of additional biotin), which would certainly be considered a complete protein and adequate in amount, gave the same growth as our casein.
2. Increasing the protein in our basal ration by increasing the percentage of our casein or of egg albumen, or by increasing the percentage of yeast, or increasing the percentage of protein by feeding soybean oil meal or linseed oil meal in place of carbohydrates, did not improve growth.
3. The work of Prof. W. C. Rose at the University of Illinois shows that the least of any amino acid necessary for the optimum growth of rats is about 20,000 micrograms daily; whereas one concentrated preparation of the growth-promoting nutrient in the liver extracts that we have used brought about optimum growth in daily doses of 20 micrograms, and another was active in daily doses as small as 2 micrograms.

It is evident, therefore, that our basal ration was not deficient in any “known” nutrient; but that this ration—and also our rations containing white flour, enriched white flour, and whole-wheat flour—lacked some still unidentified nutrient that was supplied by the liver extract and the dried skim milk. This unidentified factor is not in yeast; it would not be in white breads made from flours milled in any way or supplemented with all nutrients known to be deficient in white flour or supplemented with all known nutrients.

In our experiment we used flours. Is the unidentified nutrient so stable toward heat that if added to flour it would survive the baking of bread, or must we get this nutrient from products that do not require cooking?  It occurs in dried skim milks that have been heated to various extents.  Solutions of it, heated at various reactions for 3 hours under a steam pressure of 18 pounds, were still decidedly active, so it is quite stable.

The growths brought about by feeding various amounts of milk produced by cows fed in different ways, by feeding various samples of dried skim milks, and by feeding different kinds of cheese as supplements to our basal ration demonstrated that milk and these milk products are all good sources of the above unidentified factor.

The problem of supplementing rations deficient in this unidentified nutrient may therefore exist in the use of grains in general in either human or livestock feeding.

There has been much interest in the effect of the diet of a lactating mother upon the subsequent growth and development of her young.

We have found that our test animals at weaning are so deficient in a still unidentified nutrient that they are unable to.grow normally on a diet containing adequate amounts of all known nutrients. How did this happen? The answer is that we fed their mothers, while the young were suckling, a diet much like our basal diet, which was deficient in the unidentified factor. Under this condition the young are almost invariably deficient in the unidentified factor by the time they are weaned, and at least 99 out of 100 of them fail to eat well and to grow well when placed on our basal ration. But this condition in these young can be alleviated so that the young will eat and grow decidedly better when placed at weaning on the same basal ration. This can be done simply by supplementing the ration of their mothers with liver extract or feeding the mothers an ordinary ration while the prospective test animals are suckling. We have conducted a number of experiments along this line, and the results indicate that the growth-capacity of the young can be influenced definitely through the milk of the mother fed on our basal ration.

We have spoken only of rate of growth. What happened eventually with these rats not fed our unidentified nutrient?

Some of the pairs of young in the above experiments were continued on experiment 33 weeks, that is, until they were 37 weeks of age. At this time, the rats receiving our basal ration without this nutrient had on an average ceased to grow and weighed about 100 grams less than their sex-litter mates that got this factor. How about the functional development of the young? The young females reached the same stage of de- velopment at 34 days of age, after being on the basal ration supplemented with this nutrient for 13 days, that their litter mates attained at 54 days, after being on the unsupplemented ration for 33 days. But do the animals on the unsupplemented ration eventually reproduce all right? It appears not. They have frequently failed to conceive or have conceived and failed to litter, or have borne small litters of young that have frequently failed to survive. The significance of these results on reproduction, which have only recently been obtained, is now under investigation.

The basal ration used in all the experiments referred to contained 10 percent of fat and a moderate amount of carbohydrate. What would happen if this fat were increased to 25 or 50 percent in place of carbohydrate—would our animals still need the unidentified factor? We tried it out, and found they would need it. Then we changed the kind of carbohydrate, and fed lactose, the sugar in milk.

The growth of rats fed our basal ration with 50 percent of dried skim milk in it was normal. This diet contained about 27 percent of lactose.  But when practically this same proportion of lactose was fed in our basal ration separate from milk, the lactose depressed the growth of the rats to such an extent that it was only about 36 percent of normal. This was without our unidentified factor. With this factor in their diet, sex-litter mates grew normally, the average increment in growth due to adding this factor to the lactose-containing diet being 167 percent.

There has been considerable interest in the utilization by the rat of diets containing lactose, because it was hoped that such studies might throw light on the general function of this sugar in nutrition; but it may be that what we need to know first is how to construct a diet or prepare our test animals to conduct such experiments. Then possibly we can study the function and utilization of this sugar, which exists so universally in the milks of mammals; or we can select from the experiments of the past those which have—by chance or otherwise—met the conditions which would still make them of value.

The problems of supplying protein in the diet of humans and domestic animals, and of determining the effect of feeding high-protein diets, have received much attention. In all the experiments in which we increased the protein in the diets given rats by feeding soybean oil meal or linseed oil meal or increased amounts of yeast in place of carbohydrate, the growth of the rats has been decidedly depressed. Why? This depression of growth was not due to any quality of the rations which, irrespective of the nutrients in them, made them unpalatable. This was clear from the fact that the rate of growth of the rats fed soybean-oil and linseed-oil meal rations was increased twofold or more when they were fed a supplement of liver extract entirely separate from these rations and that the growth of rats fed the larger percentages of yeast was normal when they were fed this same supplement. In addition, the growth of young rats on the high-yeast diets without this factor was alse not depressed by increasing the yeast when enough casein was removed from their ration to compensate for the protein in the yeast. This led us to question whether excessive amounts of protein are harmful in diets when this unidentified factor is deficient in them. There is now abundant evidence that they are harmful. A diet containing an excess of protein—even though it is a good protein—in a ration deficient in our unidentified factor, fed to rats depleted of this factor, actually depresses their growth, is definitely harmful, and may even be lethal. As stated earlier, our ordinary basal ration contained 25 percent of protein (20 percent casein from which the unidentified nutrient had been removed as described above and 5 percent yeast protein). When 20 percent of coagulated egg albumen replaced the 20 percent of casein in this ration, the rats grew at the same abnormally slow rate—not much more than half of normal.

When either of these proteins was increased to 40 percent in this ration, the rate of growth was reduced to only about 25 to 30 percent of normal; and when we increased the casein to 60 percent, the rats generally died within 2 weeks. These results were obtained when their diets.did not contain the unidentified factor. When this was supplied, the growth of the sex-litter mates of the above rats when placed on the same 20-percent casein or 20-percent coagulated egg albumen diets almost doubled; the growth of those on the 40-percent casein and 40-percent coagulated egg-albumen diets almost quadrupled and tripled, respectively; and the sex-litter mates of the rats that died on the 60-percent casein Tation survived and grew at rates about 85 percent of normal.  After obtaining these results, we examined the kidneys of many of our rats that had been on the 20-percent casein diet. We observed no definite lesions, but the kidneys of all animals that had been on the diet without the: unidentified factor for considerable periods of time were actually heavier than those of their much larger sex-litter mates receiving this factor. This would suggest that there was some metabolic condition that imposed extra work on the kidneys of the rats that did not receive the unidentified factor.

In appraising the practical significance of the results reported here, one must realize that we have dealt with animals that were purposely depleted of our unidentified factor; that, although our basal ration was adequate for normal growth and development in respect to all known nutrients, it was actually deficient; and that the results that we have noted apply to test animals under these conditions.

How about humans ‘and livestock? Unquestionably the most urgent need today in the field of nutrition is a more complete knowledge of the list of constituents necessary for a complete diet. This need is emphasized by the fact that in the past each newly discovered nutrient has had a beneficial effect on our health and well being.

The experiments we have discussed disclose that we do not know all the nutrients in milk that contribute to its value in nutrition. We also do not know all the functions of the well known and recognized constituents in it.

For example: Casein is the main protein in milk. It is a peculiar protein in that it contains the element phosphorus in what has been called a “phosphopeptone" group. This same group occurs in a protein in another food, namely the vitellin of egg yolk, which like casein has evolved through the winnowing selection of ages in a food specifically adapted to the nutrition of the very young animal. For years there has been speculation regarding the function of these peculiar proteins. Recently, Ben H. Nicolet and Leo A. Shinn, of the Bureau of Dairy Industry laboratory at Beltsville, published several papers on the chemical composition and properties of these proteins, and developed an understanding of the chemical properties of this phosphopeptone group that may well form the basis for a study of its physiological function.

The war has emphasized both our ignorance and the necessity for more complete knowledge of the nutritive value of milk; fundamental work intended to broaden this knowledge is now in progress in many laboratories.

THE AUTHORS
C. A. Cary is head of the Division of Nutrition and Physiology in the Bureau of Dairy Industry.
A. M. Hartman is a chemist in the Bureau of Dairy Industry.

ALSO, IN THIS BOOK
What to Feed a Cow, by R. E. Hodgson and W. J. Sweetman, page 149.
Advances in Feeding Calves, by Henry T. Converse, page 159.
What We Eat, and Why, by Esther F. Phipard, page 753.
Protein Is Essential to Life, by D. Breese Jones, page 761.