Unidentified Substances

GEORGE M. BRIGGS.

SOME day and the day may not be far off a white-coated scientist is going to look up from his data books or his laboratory bench and tell himself, with no more excitement than his scientist's caution allows, "This is it!"

It will be a new vitamin or a new growth factor that he and perhaps many other laboratory workers have tried for a long time to find or identify exactly.

This new vitamin, or a food factor, will be a link between the first findings in nutrition years ago and today's research. It will push farther back the frontiers of man's knowledge. It will benefit people or animals or both. It will be as exciting as the solution of a mystery story and perhaps as outstanding as Dr. Jonas Salk's discovery of vaccine for poliomyelitis.

It may rank alongside the discovery of vitamins A, C, and D and the B-complex vitamins, which were complete mysteries before 1920 and minor mysteries between 1930 and 1955, when their chemical structures were discovered.

For there are still discoveries to be made of unidentified substances in foods.

Natural foods, such as milk, meat, eggs, vegetables, and cereals, contain at least several important nutrients and substances whose identity we still do not know. We call them unidentified factors. The list of them has been dwindling steadily as our knowledge has grown, but the ones remaining in 1959 seem to be important in nutrition.

How do we know that unidentified factors exist?

To find out, we start with a purified diet that contains all known nutrients in ample amounts in the form of a mixture of pure proteins, carbohydrates, vitamins, minerals, and fat. We feed it to an experimental animal and keep a record of the results. If we can get better results such as improved growth or reproduction by adding any natural food to this diet, we can be fairly certain that an unidentified factor is present in the natural food. White rats, mice, guinea pigs, chicks, and other small animals are the favorite "tool" for this work.

The next job is to identify the factor. That is not easy. We make concentrates or extracts of the factor from the natural food by chemical fractionation. We test the concentrates by adding them to the original purified diet and feeding the diet to the test animal. Thus we can follow the activity through repeated tests to the final isolation of the pure substance and to its identification. This is more difficult than it sounds and may take many months or years. There are many pitfalls and blind alleys, which can slow down the work considerably.

Often it is possible to find a 1- or 2-day test for the unknown factor if we use certain fast-growing microorganisms as the test "animals." That hastens the process. Most of the B vitamins were discovered in this way by using bacteria.

THE DISCOVERY of vitamin B₁₂ in 1948 is an example of how unidentified factors become known and identified.

A number of supposedly different unidentified factors were being studied in different laboratories before 1948.

Many of these turned out to be vitamin Bl, As far back as 1926, Dr. George Richards Minot and Dr. William Parry Murphy, of Harvard and Boston, discovered that pernicious anemia, an incurable disease in man until then, could be treated by feeding large amounts of liver. Concentrates of the antipernicious anemia factor, as it was called, became available in the form of injectable liver extracts in a few years but attempts to identify the active factor were unsuccessful.

Two groups of workers at the Agricultural Research Center at Beltsville, Md., had been working with unidentified factors for animals since the 1930's. Their investigations were along two different lines.

A number of investigators in the Poultry Division Drs. Theodore C. Byerly, H. W. Titus, H. R. Bird, A. C. Groschke, N. R. Ellis, J. C. Hammond, M. Rubin, and others studied an "animal protein factor." They learned that it occurred in protein concentrates from such sources as fishmeal and meat scraps and helped growth and reproduction of poultry. They discovered by a fortunate observation in 1946 that ordinary Cow manure is an excellent source of a similar unidentified growth factor for chicks. It became known as the cow manure factor. Long and tedious attempts to purify and identify the substance were unsuccessful, but the scientists were able to prove that the factor is synthesized by micro-organisms in the rumen.

C. A. Cary, A. M. Hartman, and their coworkers in the former Bureau of Dairy Industry of the Department of Agriculture were working at the same time on what was thought to be another unidentified factor in milk. It was necessary for the growth and reproduction of laboratory rats. This factor was different from all vitamins and minerals known at that time (1943 to 1947). They called it factor X. Many time-consuming studies were made to identify the factor, but it proved to be elusive and difficult to purify. These scientists found that antipernicious anemia factor concentrates were good sources of factor X, a finding that later proved to be useful to others (even though they believed at the time that the two factors were not identical).

In another laboratory, Lois M. Zucker and T. F. Zucker of Columbia University, a husband-and-wife team, were studying growth effects obtained by adding casein (the protein of milk), liver, or fish solubles to purified diets for rats. They obtained concentrates of an unidentified factor, which they named "zoopherin."

Other workers with rats and chicks in various laboratories, including the University of Wisconsin, Cornell University, and Lederle Laboratories, confirmed these findings and were actively attempting to identify the animal protein factor, as it was most commonly called in 1946 to 1948. All found the animal tests expensive and difficult.

Still another line of attack was being made to isolate this unidentified factor. Mary Shorb, a bacteriologist, working first at Beltsville in the former Bureau of Dairy Industry and later at the University of Maryland, discovered in 1947 that a certain bacterium, Lactobacillus lactis (Dorner), would not grow unless concentrates of the antipernicious anemia factor for humans were added to their food. She called this unknown substance the LLD factor, from the initials of the name of the bacterium.

Following this discovery, Dr. Shorb and I, at the University of Maryland Poultry Department, attempted to purify the LLD factor further. We had little success, but we were able to improve the assay enough so that it could be used by ourselves and others in short-time routine tests to identify the compound.

Using the Maryland test with bacteria, research workers in the Merck & Co. laboratories in Rahway, N. J., isolated a deep-red, crystalline compound from liver and other sources in 1948. Like others, the Merck laboratory had spent thousands of dollars and many years in attempts to find the antipernicious anemia factor. The goal was reached by the use of the 2-day bacterial test, which was better than testing each new batch on hospital patients suffering from pernicious anemia. The new compound was named vitamin B₁₂.

Thus a scientific mystery of long standing was solved in 1948, and many loose ends were brought together. After the discovery of vitamin B₁₂, scientists proved that it was not only the LLD factor but also the antipernicious anemia factor for humans, factor X, the animal protein factor, the cow manure factor, and zoopherin for chickens, pigs, and rats!

A discovery of this type leads to a large amount of experimental work and results in many benefits to mankind. It has had much practical use in animal feeding and in human nutrition and medicine. Workers in laboratories all over the world have written more than 1,500 scientific publications on vitamin B₁₂ since 1948.

Another example of a recently discovered nutrient is selenium, a trace mineral. It was an unsuspected part of an unidentified factor until 1957, when scientists discovered that compounds containing selenium had important nutritional properties in feed given rats, pigs, chickens, turkeys, and mice.

The recognition of selenium as a trace element in nutrition goes back to 1951 to the work of K. Schwarz and his coworkers at the National Institutes of Health in Bethesda, Md. They discovered that some foods, such as milk, brewer's yeast, meat, and certain cereals (corn, soybeans, and wheat) contain an unidentified substance, which they called factor 3. It prevented liver damage and death in rats fed special diets low in vitamin E and low in cystine, an amino acid.

After 6 years of intensive study and purification, Dr. Schwarz and his group in 1957 discovered that highly purified concentrates of factor 3 contained selenium. Certain crystalline salts of selenium fed in small amounts ( 0.1 part per million or less) prevented liver damage in rats. This amount is far less than the amounts necessary to produce the well-known toxic effect of selenium in feeds.

Several groups of workers discovered soon thereafter that selenium-containing compounds are also exceedingly active in preventing exudative diathesis in chicks (a condition characterized by fluid under the skin), liver damage in pigs, and heart and kidney damage in mice.

Vitamin E had to be absent from the diet before the selenium compounds were effective in all animals. Vitamin E prevented the conditions, but it was only about one five-hundredth as active as the selenium compounds. Not all the functions of vitamin E could be replaced by the selenium compounds, however.

An active search began in 1957 to find the most active selenium-containing compounds in foods and to find out why selenium-containing compounds are necessary under these conditions. As is typical of such studies, complete answers will not be found until after years of devoted work on thousands of animals and much expense.

NOBODY CAN SAY how many nutritional factors remain unknown or how soon they will be discovered.

I list some of the substances that seem to be necessary for animals and that were not identified in 1958. We have little information about their importance in human nutrition.