Carnegie Institution of Washington, Division of Plant Biology. Stanford University, California
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NIQUE reactive yellow pigments, usually found associated m t h fats, are essential constituents of the organic world. Without continual replenishment of these necessary materials by natural synthesis, few living organisms could long survive. Somewhere in a past geological age rudimentary cells produced the first of these strange yellow substances. Later, more of the remarkable colored compounds appeared, and in time elementary plants used them in new life processes. Eventually, animals feeding upon plants employed the ever-present yellow constituents for regulation and control of vital reactions in their own bodies. After eons of time man became curious about the widespread nutritional diseases that he observed in his domesticated animals and in members of his own clan. Some of these diseases were curable with special foods. Nearly 4000 years ago " . . . the liver of an ass, raw. . . " was prescribed for failure of vision in dim light. In the 19th century cod-liver oil became a popular supplemental food. But not until the 20th century were certain yellow plant pigments recognized as the primary source of a curative substance in liver. Distinguished by their preferential solubility in fats and in fat solvents, these yellow plant products have now been found in a variety of organisms. The pigments themselves occur in numerous modifications which exhibit d i e r e n t physiological activities. Recognition of the individual pigments and their complex physiological roles was dependent upon development of new physical, chemical, and biological techniaues. I t reauired careful correlation of diverse and apparently &elated obsesvations. Earliest chemical investigations of yellow fat-soluble pigments were contined to the carotene of carrots. By 1837, however, the Swedish chemist, Berzelius,'had found a similar yellow substance in autumn leaves. I t was differentfrom the familiar carotene, and he realized that it must be a new chemical compound. For
his find, he coined the term "xanthophyll" from the Greek words meaning "leaf-yellow." Discovery of xanthophyll marked the beginning of a fine distinction between two similar types of fat-soluble yellow pigments. Carotene was readily soluble in petroleum ether and was not extractable therefrom with aqueous alcohol. Xanthophyll, by contrast, was less soluble in petroleum ether and was extractable from i t with aqueous alcohol. In time, chemists correlated these dierences in solubility with the chemical composition of the pigments. I n the past 50 years some 70 or 80 xanthophylls and approximately a dozen carotenes have been isolated from plants and animals. Known collectively as carotenoids, they are the commonest yellow, orange, and red pigments in the organic world. The detection of these similar compounds in various organisms, their isolation, and the determination of their properties and chemical structures provide one of the most interesting stories in the field of modern research. This story embraces progress in practical and theoretical fields. It has been written by biologists, chemists, physicists, engineers, and farmers. I t hinges upon such basic phenomena as the nature and function of vitamins and the maintenance of life by plants. I t illustrates the complexity of relationship among agriculture, food processing, nutrition, and health. SOURCES OF XANTROPHYLLS AND CAROTENES
F i s t hints of the wide distribution of xanthophylls and carotenes in nature came in 1860 and 1864 when these yellow pigments were detected in the green parts of plants. All green plants examined thus far, ranging from the smallest unicellular diatoms to the largest trees, contain both xanthophylls and carotenes. Here these pigments occur in intimate association with the
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green chlorophyll--one part of carotene with about three parts of xanthophyll and 12 parts of chlorophyll. All these pigments, the green masking the yellow, are found within the plant cells in microscopic highly specialized bodies called "chloroplasts." Formation of the yellow carotenoid pigments in leaves often precedes synthesis of the green chlorophyll~. This is especially striking in leaves that have never been exposed to light. Young plants obtained by germination of seeds in the dark and young leaves of celery and of other blanched vegetables owe their pale yellow color to the presence of carotenoids. Upon exposure to light these immature plant parts form large quantities of chlorophyll and additional amounts of yellow pigmeuts. Xanthophylls are the principal coloring matter of specialized parts of many plants. Thus oranges, red peppers, fruit pods of the Chinese lantern, and the petals of many flowers, such as those of marigolds and the California poppy, owe their striking color chiefly to the presence of distinctive xanthophylls. Other plant parts such as carrots and tomatoes are colored by carotenes. During the early stages of their development, many of these plant parts are usually green. Later the special xanthophylls and carotenes are formed as the green pigmeuts disappear, a phenomenon observed by most housewives in the ripening of oranges, tomatoes, and peppers. Many fish, the feathers of some birds, shells of shrimps, crabs, and lobsters, and fats of many animals contain carotenoid pigments. In animals these pigments usually occur in solution in the fat or in combination with protein. Rupture of the union between protein and xanthophyll accounts for the change in color observed when crustacea are boiled. For the most part carotenoid pigments found in animals are derived from plants which are consumed as food. But absorption of xanthophylls and carotenes from the food is a highly selective process. For example, the natural coloring matter of butterfat is carotene and that of eggyolk is xanthophyll. This selection occurs even when cows and hens receive rations equally rich in both pigments.
important roles in plants. Coloration of flowers by carotenoids may serve to attract pollination insects. Curvature of plants toward light probably results from action of the light upon yellow pigments within the sensitive cells. This or a similar response may regulate the growth and development of many plants. Xanthophylls and carotenes, in conjunction with chlorophylls, help maintain the universe of living organisms. Virtually all living beings, animals as well as plants, depend upon synthetic reactions that take place in the chloroplasts. Through absorption of sunlight chloroplast pigmeuts garner the energy required for conversion of carbon dioxide and water into organic substances such as soluble sugars,starch, and cellulose. This photosynthetic process is basic to all agricultural production. It provides the primary foods of all animals as well as of man. Is the presence of this triumvirate of xanthophylls, carotenes, and chlorophylls necessary for utilization of solar energy by plants? Recegt experiments indicate that it is. Every plant that carries on photosynthesis contains these green and yellow substances. Light ahsorbed by pigments of each group is utilized in the
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FUNCTIONS OF XANTHOPHYLLS AND CAROTENES
One of the most important roles that can be ascribed to some xanthophylls and carotenes is their conversion into colorless fat-soluble vitamin A in the animal body. From certain of these carotenoid pigmeuts animals synthesize this vitamin which is essential for growth of the young and for formation of substances that render the eye sensitive to weak light. In some fishes and in some birds the yellow markings that apparently serve as protective coloration are due to xanthophylls and carotenes. Other functions are indicated by the wide distribution of carotenoids in the animal kingdom and by the preferential concentration of these pigments in tissues exhibiting great metabolic activity. Yellow fat-soluble pigments apparently play several
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photosynthetic process. Plants that lack the pigments are unable to utilize solar energy. Ancient man in his pastoral world recognized all flesh as grass. Modem man with his analytical instruments sees all flesh as sunshine, condensed by the green and yellow chloroplast pigments. COMPLEXITY OF THE NATURAL MIXTURES OF CAROTENOIDS
Mixtures of carotenoids occur in the green parts of all plants. In the past decade the existence of two leaf carotenes and of about a dozen leaf xanthophylls has been established. Leaves of all the higher plants that have been investigated contain most of these xanthophylls in approximately the same proportions. Some of the lower plants, such as bacteria and seaweeds, include mixtures of still other xanthophylls. Plants belonging to different botanical groups often do not have a single xanthophyll in common. Thus the unicellular diatoms and dinoflagellates, the two principal plants of the seas, contain mixtures of xanthophylls not found in land plants. XANTHOPWLLS OP HrOaBa L n w PLANTS m o OF rnn u m c e ~ ~ W am n e~a P ~ m r r D, I ~ T O M S ,AND D I W O ~ L A G ~ L L A ~ ~ S Neofvco-thin A Neofueoxanthin B Fucoxaothin Diadinoranfhin
Ncoperidinin Peridinin Neodinoranthin Neadiadinoranthin
Ncoxanthin Flavoranthin Violananthin Zeaxsolhin
Carotenes of the green parts of plants are subject to less variation than the xanthophylls. The principal carotene of marine and of land plants is the same, being identical with the principal beta-carotene of carrots. In some plants small quantities of additional carotenes are found. Of all the green plants examined, only in the photosynthetically active bacteria has a group of entirely different carotenes been discovered. Xanthophylls and carotenes of specialized yellow fruits are subject to tremendous variation. Some of these pigments occur in only one or two sources. Some have never been found in fruits that are still green. In a few plants these pigments of fruits represent labile structural modifications of the commonest carotenoids. ISOLATION O F XANTHOPKYLLS AND CAROTENES
Determination of the properties of organic compounds such as the carotenoids is continpent upon isolation of the substances themselves in a highl; purified state. Since carotenoid pigments occur in small quantities (from three- to four-tenths of a gram in one thousand grams of fresh leaves) and since they are very reactive, this condition has been extremely difficult to fulfill. Resolution of these natural mixtures of carotenoid pigments into their several members has been carried' out by broadening an analytical trail blazed over 30 years ago by a Russian botanist named Tswett. In its present form his unique adsorption analysis leads to preparation of pigments in a high degree of purity. It is now widely utilized by chemists for isolation, separa-
tion, and identification of many types of different compounds. The original basis of this novel and useful method was the observation that petroleum ether extracts of dried leaves form a series of green and yellow bands or zones when passed through glass tubes filled with very dry, adsorptive chalk. Separation of pigments into discrete bands on these chalk columns was enhanced by washing the adsorbed materials with fresh portions of the solvent. Isolation of pigments separated by this "chromatographic adsorption" method is a simple matter. The colored bands are loosened one by one with a long spatula and transferred to separate vessels where they are agitated with alcohol. This solvent liberates the pigment from the adsorbent. Removal of the adsorbent by filtration provides solutions of the pure pigments. Concentration of the solutions by evaporation of the solvent yields glistening orange or red crystals of the carotenoid. Successful application of the adsorption method depends upon careful selection of adsorbents and of solvents. With columns composed of a specially prepared magnesia or of lime, relatively large quantities of pure carotenes and xanthophylls have now been isolated from plants of many different species. For separation and isolation of carotenes petroleum ether is used for solution of the pigments. Separation of the xanthophylls is accomplished with a mixture of petroleum ether and acetone or with dichloroethane as solvents. Chlorophylls and certain labile algal xanthophylls are readily separable upon columns of powdered sugar. With this adsorbent, separation of the pigments is enhanced by use of petroleum ether containing about one per cent alcohol as solvent. Chromatographic adsorption is the only procedure by which many natural mixtures of the chloroplast pigments have been resolved. These labile substances, so closely related chemically that they cannot be separated by the usual analytical methods, yield readily to the selective forces in the adsorption columns. The great resolving power of the adsorption technique, now widely appreciated in other fields, has made possible purification and estimation of the pigments and determination of their physical, chemical, and physiological properties. PROPERTIES OF CAROTENOID PIGMENTS
Xanthophylls and carotenes are extremely sensitive and reactive substances. In organisms they are protected by the activities of the living cells. If the cells are killed, the pigments are destroyed by oxygen. This oxidative reaction is accelerated by enzymes and by light. In solution carotenoids are partially converted into labile interconvertible isomers by light, by gentle heat, and by iodine and other similar catalysts. They are decomposed by acids, heat, and oxygen, changing gradually to colorless products. Like the reactive filaments of incandescent lights, carotenoids must be
,
preserved in evacuated and sealed glass tubes. In nature they persist for long periods only in the cold, dark, oxygen-free ooze of the ocean floor. Results of both practical and theoretical significance have come from studies of the intense orange color of the xanthophylls and carotenes. Many careful investigations with the spectroscope have shown that each carotenoid absorbs slightly diierent proportions of blue and green light. This property, the absorption spect ~ m is, now widely used for identification of the individual pigments. It is intimately related to the arrangement of the atoms within the pigment molecules. It discloses some of the innermost secrets of chemical structure. Changes in the structure of the chromophoric portion of the molecule are accompanied by changes in the absorption spectrum. From determinations of the amount of light absorbed by the colored constituents in extracts of plants and animals the quantity of pigment contained in these sources can be found. CHEMICAL STRUCTURE O F CAROTENOIDS
Physiological as well as physical and chemical properties of the carotenoids are reflections of the arrangement of the atoms in their molecules. For example, this molecular structure determines whether or not a carotene or xanthophyll will yield vitamin A in the animal body. Knowledge of the structure also reveals the chemical relationship between carotenoids and other organic compounds. It serves as the only reliible guide in attempts to synthesize the natural pigments. In order to determine the structure of a newly discovered carotenoid, the organic chemist first ascertains the nature and the number of atoms in its molecule. His next and most difficult task is the determination of the spatial arrangement of these atoms. From a vast amount of experience he knows that molecules of organic substances are composed of a skeleton of carbon atoms linked together by bonds or units of chemical affinity. Each carbon atom has four of these bonds distributed equidistant about it. It is joined to neighboring carbon atoms by various combinations of single C-C, double C=C, or triple C k C bonds to form chains, plane rings, or complex three-dimensional structures. Chemical bonds not utilized in combinations between carbon atoms always occur in union with atoms of other elements, chiefly hydrogen and oxygen. The variety of structures that may be built from carbon atoms is enormous, but for every organic substance there is only one structure for each of its molecules. With his new carotenoid the chemist determines the number of double bonds and the number of rings of carbon atoms present in the molecule. Finally, with the aid of special reagents he cleaves the large molecule a t its vulnerable points, such as the double bonds. This process provides smaller organic molecules whose structures are usually known. This information, in conjunction with knowledge of the properties of the
pigment, makes possible deduction of the molecular structure of the original compound just as fossil remains permit reconstruction of the appearance of prehistoric organisms. As shown by chemical analysis molecules of most carotenes contain 40 atoms of carbon and 56 atoms of hydrogen. Malecules of most xanthophylls contain these same numbers of carbon and hydrogen atoms and from one to six or more atoms of oxygen in addition. These oxygen atoms render the xanthophylls relatively more soluble in alcohols than the carotenes. From an analysis of their parts all xanthophylls and carotenes were found to contain a series of carbon atoms united by alternate single and double bonds. It is these alternate single and double bonds (the so-called conjugated double bonds) that are responsible for absorption of light by carotenoid molecules. The number and the arrangement of these double bonds determine the spectral absorption properties of the pigments. The larger the number of conjugated double bonds the greater the 'absorption. Changes in the spatial arrangements of the atoms near the double bonds account for the formation of the labile isomers of both xanthophylls and carotenes. In molecules of some carotenes, as in the lycopene of tomatoes, the ends of the chain of carbon atoms are branched and contain only a few double bonds. These PIGpigments are not converted to vitamin A DIATOM SePA. in the animal body. I n the molecules of R A T E D e Y other carotenes one or both ends of the chain may be coiled into rings of carbon uM,o,po,. atoms already familiar to the chemist as DEREDSUGAR parts of the ionones, synthetic compounds having the odor of violets. Only those carotenes that contain a terminal ring identical with that in betaionone are converted into vitamin A by animals. Years of patient work in many different laboratories have demonstrated that the carbon skeleton of xanthophyll molecules is similar to that of the carotenes; hence, xanthophylls may be regarded as oxygen derivatives of carotenes. I n many xanthophylls oxygen atoms occur as 0x0 (=C=O), as cyclic oxides, or as hydroxyl groups (--C-OH) in both of the terminal rings referred to above. These pigments do not exhibit vitamin A activity. Other xanthophylls, by contrast, contain one ring free of oxygen atoms and exhibit pronounced vitamin A activity. A few carotenoids which contain acidic terminal carboxyl groups in place of the ring structures are not precursors of vitamin A. Xanthophylls found in many of the yellow parts of
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plants usually have their hydroxyl groups combined with fatty acids in the form of esters, whereas these same pigments found in the green plastids are unesterified. The solubility of these esters resembles that of the carotenes rather than that of the xanthophylls. Hydrolysis with alkali converts the esters into xanthophylls and salts of the fatty acids. On the basis of chemical structure xanthophylls and carotenes are closely related to a portion of the green chlorophyll molecule and to vitamins E and K (both found in leaves) as well as to vitamin A (not found in plants). This suggests that all these compounds may originate in nature by similar mechanisms. It illustrates how chemical investigations may uncover new and unsuspected relationships between organic materials found in living organisms.
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