1938: Biological Chemistry

Fields of Research.

Chemistry in relation to biology, or biochemistry, overlaps frequently other fields of scientific study, such as agriculture, medicine, physiology, and bacteriology. The major fields of research in biological chemistry can be satisfactorily viewed, however, in terms of the primary purposes involved: (a) to gain a clearer insight into the structural nature and composition of living cells; (b) to follow the chemical changes that characterize the life processes of each type of tissue; and (c) to study in a fundamental manner the problems of growth, nutrition, health and disease. Although progress has continued steadily in each of these three fields for a number of years, only very incomplete concepts are yet available concerning the composition and function of the simplest cells.

Structure and Composition.

Proteins, which constitute the dominant part of the active cell mass, have been studied extensively and continuously in many laboratories. The most important recent advances in unraveling their make-up have been in relation to their particle size, their structure when oriented in films and crystals, and the exact mode of linkage of their structural units, the amino acids. Bergmann's work has been of special interest in establishing the regularity of the sequence of amino acids in the long, chain-like structure containing approximately 288 individual unit parts. The larger protein particles appear to be built up as multiples of the 288 unit particles, in agreement with the earlier findings of Svedberg that protein generally had molecular weights of 34,500 or multiples of that number.

The repeated isolation and identification of huge protein molecules, that occur as filterable viruses and bacteriophages, by Stanley, Northrop, and Wyckoff have led to a new phase of protein studies. The molecular weights of crystalline virus proteins have been reported as approximately 20,000,000, and Wyckoff has reported values as high as 300,000,000 for the molecular weight of bacteriophages. The particles appear to possess autocatalytic power, producing increasing amounts of like protein material from the cellular constituents of bacteria or host tissue, as in tobacco-mosaic. Particles of the nature of viruses and bacteriophages represent the nearest approach to bridging the gap between living and non-living material. The possibility remains that bacteriophages represent true portions of living matter rather than pure crystalline proteins in the usual sense of referring to a pure compound. The presence of nucleic acid in the bacteriophages provides preliminary evidence of the presence of nuclear portions of cell structure.

The detailed chemical structures of starches, cellulose, and the simpler sugars have been established much more clearly than the nature of the proteins. Hence, recent progress in the study of the carbohydrates has been less striking, and characterized by gradual progress rather than by major advances.

The study of oils and fats has progressed steadily in regard to (a) the nature of the non-soap-forming fraction, and (b) the distribution of fatty acids in accordance with biological classification. Searching for vitamins and hormones in the unsaponifiable matter of fatty materials has led to the realization that hydrocarbons, alcohols, ethers, esters, ketones, and a great variety of physiologically important types of compounds are normally present in nearly all natural fats. In only a few cases has it been possible to assign functional activity to the non-fatty-acid portion of fats, but the recent trends of investigation point toward an increasing recognition of the importance of such compounds in both plant and animal physiology. The fact that compounds in this class of materials (unsaponifiable matter) furnish vitamins A, D, E, and K, the cortical hormones and the sex hormones, may serve to illustrate their importance.

The problem of molecular structure in relation to the fatty acids in oils and fats has been studied chiefly by Hilditch and associates, who have been able to correlate a number of chemical characteristics with biological classifications in both animals and plants.

An accomplishment of unusual interest was the preparation of optically active fats by H. O. L. Fischer and associates. Esters of glycerol with fatty acids and phosphoric acid were prepared from the optically active glyceric aldehydes, which were, in turn, prepared from d- and l- forms of a common sugar, mannose.

The catalysts (enzymes) which control the rate of nearly all chemical reactions in living cells are now known to be essentially proteins. The pioneering work of American investigators in proving the protein nature of enzymes, especially the work of Sumner, Sherman, and Northrop, long in disagreement with the German research school headed by Willstaetter, has been fully confirmed. Many of the enzymes have been obtained in pure, crystalline form, and in every case they have proved to be proteins. The digestive enzymes and a number of others have not shown any evidence of the protein being linked to a special, catalytically-active group; but the enzymes that control the combustion processes in the body have been found, in a number of cases, to have a special (prosthetic) group attached to a native protein. Both parts of the molecule are essential to its normal rôle. Metallic elements such as copper and iron, and organic groups such as the vitamins have been identified as essential parts of enzymes. The native catalysts can be broken apart and again synthesized without loss in their catalytic activity or any apparent change in their properties. Copper is found in at least two such protein compounds in plants, and occurs in still other protein combinations in animals. More and more it becomes apparent that the remarkable effects of minute traces of many of the elements, the hormones, and the vitamins in the animal body, are due to their rôle as catalysts after being linked to specific proteins.

Although a knowledge of the nature of enzymes is basic to an understanding of even the simpler changes in all living cells, relatively few direct applications have been found for them in agriculture or medicine. Recent work has made use of the characteristics of one of the enzymes in milk, to provide the basis for a valuable and practical test for adequate pasteurization of milk. The heat treatment of milk that has proved to be adequate for making it safe from transmission of disease is also just sufficient to destroy the enzyme in milk that acts upon phosphate esters (phosphatase). Both low-temperature (142° F.) and high-temperature (160° F.) pasteurization of milk destroy the enzyme to a degree that makes the test of practical value for detecting underpasteurization.

Chemical Changes within the Body.

The use of 'tracer' elements to 'tag' foodstuffs, and thus to provide a means of following their course within the various parts of the body, has proved to be a very valuable technique. The work of Schoenheimer, Krogh, and others, using heavy hydrogen (deuterium) attached to fatty acids, furnished considerable insight into the changes that the fats undergo in the animal body. The study of nitrogen (and indirectly protein) metabolism has been undertaken in a similar manner by Schoenheimer and others using heavy nitrogen. Although the quantities of the heavy forms (isotopes) of the elements can be determined very accurately by analysis, their chemical reactions are essentially the same as the common forms of the elements, thus providing a means of tracing the course of a given fat or protein through successive stages of alteration within the different organs and tissues of the body.

More recently radioactive phosphorus has been used by Hevesy and Artom to study many phases of intermediate changes in fats and phosphates in biological processes. Lawrence and associates at the University of California have also launched a program of studies with radioactive elements in plant and animal cells. It is interesting to note how quickly the research tools of the physicists and physical chemists have been adapted to the study of physiological problems by those working in biochemistry.

Vitamins.

The general term 'vitamins,' to represent organic (carbon) compounds that are required in minute amounts in the food supply of animals, is still useful in both technical and lay literature. The use of a letter (A, B, C, or other) to represent each different vitamin has become less necessary, however, for several reasons. The number of such factors covered formerly by a single letter has made the use of letters often confusing and awkward. The former vitamin B₂, for example, included many different compounds with entirely independent functions and properties. The extensions of subscripts to B₃, B₄, B₅, B₆, etc., and the parallel introduction of additional letters, G, H, etc., to cover the same series of newly-identified vitamins led to considerable confusion. In addition, the work of Bills, Hicks, and others has shown that ten or more different compounds can all serve independently as 'vitamin D' in the animal body. The response, in terms of protective dosage, varies for the different forms of 'vitamin D,' depending upon whether the rat's, chicken's, or human's response is measured. In a similar sense, the vitamin A requirement can be provided for by feeding any one of six or more different compounds that are closely related in molecular structure. Hence as each vitamin-like substance becomes clearly identified chemically and physiologically, there is a great gain in clarity of expression when specific chemical names are used for each particular substance under discussion. In accordance with this trend toward chemical nomenclature, vitamin B₁ is called thiamin; one of the fractions from the old B₂ is riboflavin; and another (G) is nicotinic acid; and vitamin C is generally called ascorbic acid.

Recent progress in the study of vitamin A has been of special significance in relation to its formation of a series of colored protein compounds, visual purple, visual yellow, etc., in the retinal rods of the eye. The compounds are sensitive to light, undergoing a series of chemical changes that constitute an important part of the functioning of the eye. Attempts to test the rate of sight-recovery after exposure to a bright light for a clinical indication of the relative degrees of vitamin A deficiency have met with varied degrees of success. Some investigators have felt that there was a good correlation between the recorded time period of sight readjustment and state of nutrition, but others have observed very poor correlation between the test results and the apparent vitamin A intake. In addition to the vitamin A compounds observed in eye tissue, Hicks has brought out further evidence, by high-vacuum distillations, that vitamin A exists in fish-liver oils in a number of different ester-type combinations with fatty acids. High-vacuum distillations from fish-liver oils also demonstrated the occurrence there of a series of natural compounds having the common property of serving as vitamin D in the animal body.

Vitamin E, the third fat-soluble vitamin to be identified, has been synthesized recently, in the course of proving its exact molecular structure. There is still no generally accepted evidence of its value in clinical practice, although it is clearly essential for normal reproduction and protection from muscular dystrophy in rats. It also has the capacity to serve as an antioxidant or anti-rancidity agent in vegetable oils.

The newest fat-soluble vitamin is K, first identified as an anti-hemorrhagic factor for chicks. It is widely distributed in green leafy foods and may be found in animal fats. Cod-liver oil is deficient in the factor, and most cereals are deficient. Even though it has not been possible to demonstrate vitamin K deficiencies in humans or small experimental animals such as rats and guinea pigs, clinical usage of the vitamin has been reported for the purpose of promoting blood clotting. Delayed clotting time due to obstructive jaundice appears to respond favorably to vitamin K therapy, thus making the apparently non-essential vitamin a useful tool in surgery. Isolation of the crystalline vitamin has been reported, but its molecular structure has not been established.

The original vitamin B group has been extended to include at least five and probably eight or more factors. Of these, the molecular structures of thiamin (B₁), riboflavin (B₂), and nicotinic acid (G or B₂) have been clearly demonstrated, and syntheses on a commercial scale have been developed. During the past year vitamin B₆ was isolated as a pure compound, but its complete structure was not established.

The function of vitamin B₆ was shown to be related to the utilization fats in the animal body and to the protection of rats against a peculiar type of skin lesion. The latter was most marked about the paws, eyes, and mouth.

Late in 1937, Elvehjem and associates at the University of Wisconsin gave a preliminary report of the isolation and identification of nicotinic acid and its amide as the anti-black tongue factor for dogs. They also pointed out the probability that the same compound would cure human pellagra. Within a very short time Spies and others at the University of Cincinnati reported the successful cure of clinical pellagra, probably America's most damaging deficiency disease, by the use of nicotinic acid. This finding provided a beautiful illustration of the way in which a research finding with small laboratory animals under carefully controlled conditions may be utilized for the improvement of human health on a large scale. Detailed reports of the work at the University of Wisconsin and in a number of medical centers appeared during 1938. Nicotinic acid (as its amide derivative) had been identified earlier as a growth stimulant for some of the lower forms of plant life and as a catalytic agent in the burning of foodstuffs in the animal body. The vitamin apparently combines with phosphoric acid and specific proteins in living plant and animal cells to form one of the chief catalysts or 'carrier agents' concerned with combustion types of reaction. The primary action of the vitamin appears to be in removing two hydrogen atoms at a time from such food stuffs as sugars, and transferring them to other systems that finally carry them to oxygen breathed in from the air.

Vitamin B₁ is concerned primarily with splitting off carbon dioxide from foods being burned in the body. The Wisconsin group showed during the past year that the nerve degeneration, or polyneuritis (clinical beriberi) that is usually associated with a deficiency of vitamin B₁, is in reality a side effect, due essentially to other factors than a simple deficiency of B₁. Alcoholic polyneuritis, characterized by a deficiency of vitamin B₁, was shown to be fairly common in the United States and European countries. Careful studies with plants during the past year brought out a number of interesting facts regarding the growth stimulating effects of the vitamin and the variation in capacity of different plants for synthesizing the vitamin from simpler materials. Bonner and Robbins have been particularly active in studying the rôle of the water-soluble vitamins in plants.

Riboflavin, often referred to as vitamin B₂ in the older literature, has been shown to serve as another of the major 'carrier agents' in the combustion reactions within animal and plant cells. Like nicotinic acid and thiamin, it combines with phosphoric acid and specific proteins to serve as a catalyst.

Vitamin C synthesis can be accomplished by practically all plants and animals other than man, monkeys, and guinea pigs. The rôle of the vitamin in a chemical sense has not been established clearly. Work in the author's laboratory has shown that the vitamin probably does not function as a major 'carrier agent' in a manner similar to the rôle of the above B-complex factors. An entirely new relationship of the vitamin to the metabolism of volatile fatty substances in rats was shown during the year. In another laboratory its function as a plant growth stimulant was clearly demonstrated.

Chemotherapy.

Clinical success with the use of sulfanilamide-type compounds in combating such infections as pneumonia, streptococcic sore throat, gonorrhea, and streptococcic meningitis has stimulated an intensive extension of the work of chemists in the search for more effective compounds with less risk of injury in the course of treatment. Of the new types of compounds related to sulfanilamide, sulfapyridine has given the greatest promise, particularly in the treatment of pneumonia.

An entirely independent research program by Cretcher and associates at the Mellon Institute, in collaboration with Dr. Maclachlan and associates for the clinical work, has developed a promising antipneumococcic agent that is derived from the cinchona alkaloids (a series that is closely related to quinine). Their product is less toxic than the sulfanilamide or sulfapyridine types of compounds and is highly protective.