The Discovery of Vitamins and Coenzymes

As early as 1750 it was recognized that green vegetables and citrus fruits could prevent the dread disease scurvy, which afflicted ancient sea voyagers, causing hemorrhages of skin, gums, and joints, followed by death. At about that time, Captain James Cook showed that sailors could avoid scurvy during long voyages by eating local green vegetables and grasses. Soon thereafter British seamen became “limeys.” The chemical structure of the active component, vitamin C (Fig. 1), was established in 1933. In a similar way, from about 1830 cod liver oil was used to prevent rickets. The active ingredient was vitamin D (Fig. 1). Later, vitamin A (Fig. 1), also present in cod liver oil, was recognized for its prevention of night blindness and maintenance of healthy skin. In oriental countries the disease beriberi, with its strange paralysis called polyneuritis, killed millions. A persuasive demonstration that this was a deficiency disease came in 1893 when Eijkman, working in Indonesia, demonstrated that chicks fed the white rice consumed by the local populace developed a rapidly fatal paralysis. However, the chicks could be completely cured by prompt feeding of a rice bran extract. It was 1926 before the curative compound was isolated from rice bran, characterized chemically, and named thiamin (Fig. 2). In 1912 the Polish biochemist Casimir Funk proposed that the four diseases scurvy, beriberi, pellagra, and rickets resulted from the dietary deficiency of vital nutrients which he imagined to be amines. He called them vitamines.

Figure 1 The structures of vitamins A, C, and D.

Figure 2 The vitamin thiamin and its coenzyme form thiamin diphosphate (thiamin pyrophosphate).

At about the same time, McCollum and Davis and others discovered that rats fed on semi-artificial diets required small amounts of “accessory growth factors.” Growth of rats required both a fat-soluble material A and a watersoluble material B. Factor A, which could be found in milk, was later shown to consist of what we now call vitamins A, D, and E (Fig. 3). In 1939 another essential fat-soluble nutrient, vitamin K (Fig. 3), was isolated from plant sources. It was designated K for koagulation, because it was needed for blood clotting. The water-soluble factor B cured beriberi in chicks. However, it was also shown to consist of several components, and could better be described as a B complex. The beriberi curative factor thiamin, which was easily destroyed by heat, was designated B₁. Another nutritionally essential component, B₂, was stable to heat. The major growth-stimulating component of B₂, the yellow fluorescent compound riboflavin (Fig.4), was designated vitamin B₂. Other water-soluble components were identified using a variety of tests. Nicotinamide was found in the coenzyme nicotinamide adenine dinucleotide (NAD; see Fig.8) in 1935. Thecorresponding carboxylic acid nicotinic acid (also called niacin, Fig. 4) is also an active vitamin. It was a well-known compound that had been prepared in 1867 by oxidation of nicotine, but had not been recognized as a nutrient until 1935 when it was shown to cure “black tongue” of dogs and shortly thereafter human pellagra. Biotin (Fig. 4) was first identified as a growth factor for yeast. Pantothenic acid (Fig. 4), which is present in plentiful amounts in many foods, was recognized as a curative agent for a dermatitis of chicks. Vitamin B6 (Fig. 5) prevented a facial dermatitis in rats (“rat pellagra”). Folic acid, first described by Lucy Wills in Bombay, was identified as a vitamin that cured a macrocytic anemia that was often associated with pregnancy.

Figure 3 Structures of vitamins K and E and of the related ubiquinone (coenzyme Q) and plastoquinone.

The curative material, which is abundant in green leafy vegetables, was named folic acid. However, this name is usually reserved for the synthetic compound used in dietary supplementation. The natural forms are largely the coenzymes (Fig. 6), which are collectively called folates. The last of the accepted human vitamins to be discovered was vitamin B₁₂. A cobalt-containing organic compound needed in very small amounts, it cures and prevents pernicious anemia, which was often a fatal disease of people over 60 years of age. Its complex structure (Fig. 7) was determined by X-ray diffraction after numerous efforts at chemical characterization had failed. However, cyanocobalamin, thecompoundisolatedandtheformused in nutritional supplementation, is an artifact of the isolation and synthesis. The natural vitamin may have OH in place of CN but consists largely of the coenzyme forms.

Figure 4 Four components of the vitamin B complex: riboflavin, nicotinamide, biotin, pantothenic acid, and the acyl carrier carnitine. Thiamin (vitamin B1, Fig. 2) is also a member of the B complex. Carnitine, which has an essential acyl carrier function in the human body, is a vitamin for flour beetles.

Figure 5 The vitamin B6 (pyridoxine) family: pyridoxol, pyridoxal, pyridoxamine, and the coenzyme forms pyridoxal and pyridoxamine phosphates.

Have all of the vitamins been discovered? Rodents have been reared on almost completely synthetic diets. However, good health in human beings may require additional materials. For example, some essential compounds might be made by intestinal bacteria. Some essential coenzymes such as lipoic acid, ubiquinone (coenzyme Q), and pyrroloquinoline quinone (PQQ) may be vitamin-like. Their presence in foods may be beneficial. Some individuals may need as dietary components these compounds, which are normally made by the body. Another example is the acyl-carrier molecule carnitine (Fig. 4), which is a growth factor needed by a common species of flour beetle, but is synthesized in adequate amounts by most human individuals. However, a few children require dietary carnitine. Inositol is an essential growth factor for yeast and is sometimes regarded as a vitamin. Flavonoid compounds, abundant in citrus fruit and other plant sources, have also sometimes been classified as a vitamin. Several amino acids, common constituents of proteins, must also be present in the human diet. These are needed in relatively large amounts and are not classified as vitamins. The role of small amounts of another amino acid taurine, which is essential for cats, in human nutrition is of current interest. The essential omega-3 and omega-6 fatty acids are also necessary dietary constituents, as are numerous metallic elements.

Figure 6 The coenzyme tetrahydrofolic acid (tetrahydropteroylglutamic acid). The vitamin folic acid has two additional double bonds (dashed) in the second ring. Most of this coenzyme exists within cells as more complex forms containing additional glutamic acid units attached to the side chain at the upper right.

The discovery of coenzymes and catalytic prosthetic groups came in part from biochemical studies of yeast, of alcoholic fermentation, and of respiration. Buchner in 1899, discovered that a cell-free juice prepared from freshly ground active yeast cells still fermented sugar. Pasteur and others had previously maintained that fermentation required intact cells. Dialysis of the yeast juice stopped the fermentation, but the material that diffused out, which was called cozymase, could be added back with restoration of the fermentation ability of the juice. Cozymase was soon found to consist of a mixture of the nicotinamide-containing compound now called NAD (Fig. 8), magnesium ions, and thiamin diphosphate (Fig. 2). NAD was quickly recognized as a component of a “respiratory chain” in animal and yeast cells. Since about 1910 this chain has been recognized as beginning with the hemoglobin-like oxygen-binding protein cytochrome oxidase and an intensely yellow flavin compound that was identified as the riboflavin derivative FAD (Fig. 9). The cooperative functioning of these vitamin-containing compounds, together with associated proteins, established the concept of coenzymes. Additional compounds, isolated from natural materials, containing vitamin B₆, pantothenic is a growth factor needed by a common species of flour beetle, but is synthesized in adequate amounts by most human individuals. However, a few children require dietary carnitine. Inositol is an essential growth factor for yeast and is sometimes regarded as a vitamin. Flavonoid compounds, abundant in citrus fruit and other plant sources, have also sometimes been classified as a vitamin. Several amino acids, common constituents of proteins, must also be present in the human diet. These are needed in relatively large amounts and are not classified as vitamins. The role of small amounts of another amino acid taurine, which is essential for cats, in human nutrition is of current interest. The essential omega-3 and omega-6 fatty acids are also necessary dietary constituents, as are numerous metallic elements.

Figure 7 Vitamin B12 (cobalamin), the first discovered carbon-cobalt compound and its coenzyme forms methylcobalamin and 5´-deoxyribosylcobalamin.

The discovery of coenzymes and catalytic prosthetic groups came in part from biochemical studies of yeast, of alcoholic fermentation, and of respiration. Buchner in 1899, discovered that a cell-free juice prepared from freshly ground active yeast cells still fermented sugar. Pasteur and others had previously maintained that fermentation required intact cells. Dialysis of the yeast juice stopped the fermentation, but the material that diffused out, which was called cozymase, could be added back with restoration of the fermentation ability of the juice. Cozymase was soon found to consist of a mixture of the nicotinamide-containing compound now called NAD (Fig. 8), magnesium ions, and thiamin diphosphate (Fig. 2). NAD was quickly recognized as a component of a “respiratory chain” in animal and yeast cells. Since about 1910 this chain has been recognized as beginning with the hemoglobin-like oxygen-binding protein cytochrome oxidase and an intensely yellow flavin compound that was identified as the riboflavin derivative FAD (Fig. 9). The cooperative functioning of these vitamin-containing compounds, together with associated proteins, established the concept of coenzymes. Additional compounds, isolated from natural materials, containing vitaminB6, pantothenic acid (coenzyme A, Fig. 10), folic acid, and vitamin B₁₂ are among many substances that are now described as coenzymes.

Figure 8 The nicotinamide-containing coenzymes nicotinamide-adenine dinucleotide (NAD) and nicotinamideadenine dinucleotide phosphate (NADP). Also illustrated are their biological functions as hydrogen carriers. The nicotinamide ring accepts a hydride ion (H−) transferred directly from a substrate molecule.

NAD, NADP, and thiamin diphosphate were found to bind reversibly to their host proteins. NAD and NADP, as their reduced (NADH, NADPH) and oxidized (NAD⁺, NADP⁺) forms (Fig. 8), were found to act as hydrogen carriers, moving freely between two or more catalytic proteins. In contrast, FAD and pyridoxal phosphate (PLP, Fig. 5) are extremely tightly bound to some proteins and normally function without dissociation from the catalytic protein. Still others, such as biotin, are covalently bonded to proteins (Fig. 11). The same is true of some FAD derivatives. These tightly bound cocatalysts are often referred to as prosthetic groups. These include a great variety of both organic and metallo-organic structures. Among the latter are the heme proteins. Vitamin C (ascorbic acid or ascorbate; Fig. 1) is unusual in functioning largely in a free, unbound form, and often at a very high concentration. This is also consistent with its high nutritional requirement for human beings. Vitamin A has a special role in vision. The aldehyde retinal (Fig. 1) combines with proteins of the retina to form the light receptors of the visual cells. Vitamin K has a specialized function in formation of a series of proteins needed for blood clotting. Both vitamin A (as retinoic acid) and vitamin D (as hydroxylated derivatives) serve as important hormones.

Figure 9 The coenzyme forms of riboflavin, riboflavin 5´-phosphate (FMN) and flavin-adenine dinucleotide (FAD).

Figure 10 Coenzyme A and its constituent components, which include the vitamin pantothenic acid.