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The cartenoid pigments of British Columbia pilchard oil Eastham, Arthur Middleton 1939

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ft it fl'S i r 2. C •> THE CAROTENOID PIGMENTS OF BRITISH COLUMBIA PILCHARD OIL. A Thesis submitted i n Partial Fulfillment of tlie Requirements for the Degree of Master of Arts at the University of British 'Columbia by Arthur M. Eastham and Kenneth A. West April 1939 ACKNOWIJEDGEBENT The authors wish to thank Dr. R . H . Clark for hxs kind assistance and the many helpful suggestions given throughout the work. £LZL,3 O ?l% W2* c J 1 T a j n e r s o f Western Chemical-Industries i/ca. ,±or hxs interest and generous supply of Pilchard G i l , ^ * Since the pigments of plants have always attracted the attention of man, i t is not surprising to find that they have been subjected to a great deal of scientific investigation. The fascinating changes of colour which take place in the plant during the spring and f a l l , and the beautiful colours of flowers and fruits at maturity have undoubted-l y stimulated much of this research, but i4 is significant, that very few problems are ever very thoroughly attacked unless they are of in-dustrial or physiological interest. Erom earliest times, man has used paints and dyes and until the modern era nature had to be the source of such coloring materials. When however, with the development of organic chemistry, i t became possible to synthesize natural materials, the synthesis of colouring matters was not overlooked, and since the f i r s t step in such synthesis is the determination of the structural formulae, i t i s not surprising to find that the structures of many of the natural pigments have been known for a considerable period of time. The physiological aspect of pigmentation is of more recent origin, but nevertheless i t has been perhaps the greatest single stimulus to the study of the plant pigments which we have had. It might be said that, on the basis of distribution, there are three main classes of plant pigments, namely the anthocyanins, the chloro-phylls and the carotenoids, and of these at least two, the chloro-phylls and the carotenoids are of distinct physiological importance. Probably the most outstanding of a l l the processes carried on by c • living material is photosynthesis, the process by which organic compounds are formed in the plant from carbon dioxide and water, and a process which is completely dependent on the green chloro-phyll pigments, The carotenoids, which until recently have under-gone but l i t t l e investigation, have recently with the discovery . of their close relationships to vitamin A , come very much to the fore and i t is doubtful i f any single field of biochemistry -ssss has received much more concentrated attention over a period of five or six years. The three main classes of pigments have chemically and physiologically, but l i t t l e in common. The chlorophylls, we know from the brilliant researches of Willstatter and his students and more recently of H, Fischer and his coworkers, to have the follow-ing structure: J) ^ j ^ - c W , c V , r — T A V • • -!T7===eJ/ — ! ! . J U The carotenoids, some of which, for reasons yet unknown, are always found associated with the chlorophylls in the plastids, are hydrocarbons, or derivatives of hydrocarbons, which consist of long chains of carbon atoms joined by a series of conjugated double bonds. The anthocyanins, and we may include here . for the moment -3-the related pigments, xanthones, flavones and madder reds, are aromatic compounds and have as the base for their molecules, such compounds as benzene, napthalene and anthracene. They are, unlike the other two groups, chiefly water soluble, and are responsible for most of the blues and violets as well as some of the reds and yellows found in nature. The natural carotenoids are a l l yellow to red in colour, and unlike the.other groups, are not confined to plants although plants seem to be their original source. As yet there i s no evidence of animals having synthesized any carot-enoids ^ although they unquestionably do change plant carotenoids to typically animal carotenoids such as astacin, which is found in so many of the lower animal forms. As has been mentioned above?the physiological importance of the group has been one of the reasons for tlie recent rapid devel-opment of our knowledge of the carotenoids. Another factor, however, which has influenced the development of the f i e l d is the development of methods of investigation. The great di f f i c u l t i e s in handling these pigments compared to those of,: for example the anthoeyanins, have had a definite retarding effect upon the accumulation of accur-ate data, but i t is interesting to note that some of these new methods when now applied to the supposedly thoroughly investigated anthocyanin group suggest that these pigments might be advantageously reinvestigated in the light of our newer knowledge. It would be well perhaps, before going any further, to consider the chemical and physical properties of carotenoidsj and the methods . which are used in their separation and identification. A carotenoid may be defined as a nitrogen-free polyene pigment consisting wholly or chiefly of a long acyclic chain of carbon atoms united in an uniterrupted sequence of conjugated double bonds. The colour varies from bright yellow to deep red but may even be violet or dark blue. In general,the depth of shade increases with the number of consecutive conjugated double bonds. The existing nomenclature of these, compounds is unfortunately rather confusing. Due to the presence in the literature of large numbers of historical names for the compounds of this group, attempts at standardizing the naming of the pigments has met with only partial success. The basic hydrocarbons which were formerly known under the one name "carotene" may now be divided into at least six compounds, all. of which are characteristically soluble in benzine and insoluble in ether, and a l l of which have the general formula G40H55. In naming these compounds i t has become common practice to c a l l the principle members of the hydrocarbon group "carotenes" to conform with typical hydrocarbon nomenclature. There are recognized at present four carotenes, designated y <*>- , An isomer of these, which is of considerable importance is lycopene, the pig-ment of ripe tomatoes. Irom these basic hydrocarbons are probably derived the oxygen-ated carotenoids, which may have as their substituents, the groups -OH^tC - Gs-O)6)H,-CH0 & OGHg. Formerly they were a l l classed as "xanthopllylB'-jalthough some authors preferred to use this term for pigments containing hydroxyl groups only.. An attempt is now being made to c a l l the hydroxy-carotenoids, carotinols to con-form with the international system of alcohol nomenclature and the name "xanthophyll" would then be reserved for the 3.3A dihydroxy-a-carotene, which is so common in green plants^ and which sp es also by the name of lutein. Under this system, the name "carotenone" should be applied to the keto-derivatives of the hydrocarbons, but i t has unfortunately been already applied to other compounds and is therefore not avail-able. The numbering systems used in naming carotinoids are several, but throughout this review, the system of Karrer w i l l be adopted. It is illustrated in the following examples: a ^ cJ* CJ3 / \ . <r*3 ft .CJ,y Cx CJf, at* JJZC C-CJi*CJi-C*CM-Ck*CJi-C-CJi*CJr-CA-CJl~C-CA~C&-U^ '(J, \ V JY Cj/j cJ^t % Y 3.3'- <&/>i/cfroxu-^ - Csroieve NCjf -6-In nature the fomrty carbon atom, or G40 carotenoids^are most common and w i l l therefore be considered here almost entirely. Formula 1 above represents the structure of lycopene. It is a straight chain compound in which the /3-ionone rings at each end are unclosed, p. carot&ne (Formula II)has the same structure with the exception that both /S-ionone rings are closed. Y- carotene differs from these two pigments in having only one of the rings closed and (pC- carotene, although having both rings closed, has a shift in the position of the double bond of one ring from 5.6 to 4*5, /3-Carotene shows strong vitamin A activity,x*ir- said carotenes less. The formula of Earrer for the vitamin A molecule is here given for purposes of comparison Q ' Ch3 Cit3 The- close relationship between this and the vitamin A active carotenoids i s obvious. If j3- carotene were hydrolysed between the central (15*15) carbon atoms to produce two primary alcohols, two molecules of vitamin A would be formed. In the case of a-and "V- carotenes i t i s found that the vitamin A activity is only half that of <fi- carotene and a glance at the formulae w i l l show the reason. In a-and > — carotene only one of the /<?-ionone rings i s identical with that of vitamin A, the difference in the other ring being one case a shift in the double bond and in the other, an unclosed ring. Cryptoxamthin, having the structure 3-hydroxy- --carotene i s also active as a source of the vitamin, but Z*a»anthin, 3,3A -dihydroxy-/? -carotene is inactive showing that the presence of a hydroxy1 group, in the 3-position of the /3 -ionone ring at "least, is sufficient to destroy the activity. The methods of handling,, detecting, separating and identifying pigments of the carotenoid group are not numberous. The compounds are very sensitive to physical conditions especially in solution. They are usually destroyed by temperatures of above 55 degrees C and are sensitive to oxidation under atmospheric pressures. When-ever possible, especially in concentrated solutions, they should be handled under nitrogen. Probably the most important tool in the hands of those studying carotenoids i s the method of Tswett chromatographic analysis. It has been well described in the literature,but the review by Cook (i ) would be worth reading hy those interested. The method is based upon adsorption of the pigments from sol-ution by chemical compounds such as alumina, magnesia, calcium hydroxide or calcium carbonate. A glass tube of desired dimensions is connected with a.suction flask and then packed tightly and evenly with the adsorbent,which may be mixed with a f i l t e r - a i d of some inert material to increase the rate of flow. A solution of the pigment or pigments i s then introduced at the top of the column and allowed to percolate down. This solution for best adsorbtion should be f a i r l y concentrated and should be made up in a solvent in which the pigment , , -8-tfor the xanthophylls,benzine is often satisfactory, but carbon disulphide and ether are often used. The pigments are adsorbed on the column in zones which may be sharp or quite diffuse. These zones or bands are then sharpened by washing with fresh solvent and are then usually cut apart mechanically to separate them. The pigment separation thus obtained i s very satisfactory. In the case of Petalozanthin, which- w i l l ho-dioouoaod elsewhere, a new pigment was discovered by means of chromatogrphic analysis. When a l l other methods of separation had indicated Petaloxanthin to be the same as Antheraxanthin, a mixture of the two pigments showed two bands on the column. It can also be noted here that this method has been applied to anthocyanins and has suggested that some of the earlier work in this f i e l d might be profitably reinvestigated. Karrer applied the method to the purification of vitamin A and through its aid was able to finally isolate this compound, from the unsaponifiable res-idue of fish liver o i l , in a pure crystalline state* The only disadvantage of the method i s the possibility of isomerization of the pigments in the process ( 2 , 3 . ) „ Por this reason i t i s important to choose the adsorbent to be used with some care. Charcoal has many disadvantages; i t is too strong an adsorbent and makes elution of the pigment d i f f i c u l t , i t is an oxidizing agent and can produce decomposition of the pigment, and fina l l y i t s colour makes i t impossible to see the bands. A rather strong adsorbent which does not cause much decomposition is alumina which can be obtained in various degrees of activation, strain (4). found the moat suitable adsorbent for the carotenes to be a specially-prepared magnesium oxide* Bather closely related to the chromatographic analysis method, is a method of detecting mixtures which has been used with some success and which goes by the name of "capillary analysis". When a strip of fi l t e r paper is placed with one end in a solution of pigments, i t is found that the solvent rises in the f i l t e r paper and the pigments form definite 2ones on the paper. The relative position of the bands together with their breadth and colour, ane characteristic of certain pigments. Kyiin (s ) in his investigations of the carotenoids of higher plants and algae used this method extensively. His results however, are not sufficiently accurate or fundamental to allow their interpretation in terras of our present knowledge and i t can be said that this method is very limited in its application. Tying with the chromatograph in value to the investigator is the spectroscope. By means of these two tools he can give almost positive Identification to a pigment although i t is usual not to consider identification complete and unquestionable unless the pigment is obtained in pure crystalline form, so that melting point determinations and analyses can be made upon i t . However, because of the very small amounts of pigment that may be available and because only small amounts are required for spectroscopic examination, this method has been of. inestimable value. -10-The absorbtion curves of nearly a l l the pigments have been accurately worked out and are recorded in the literature. It is usual now to record simply the maxima of the absorbtion bands and the solvent in which they are measured. Much of the early ?/ork has been of l i t t l e value because the workers have failed to record important data such as solvent employed. The solvent used is of particular importance because i t can cause shifts of at least 40 mu in the pos-ition of any one band* Solubility tests constitute the fi n a l important method in the identification of carotenoid pigments,, The so called Kuhn and Brockmann micro method is essentially the distribution of pigments between two mutually immiscible solvents, and of these themost Important distribution is that between petroleum ether and aqueous methanol. By use of this technique, the basic hydrocarbons and «wW cant* sepawieJ ^ ^ ^ o*$s««sW <^™i>f7ot&r pigment waxes (carotenoids^because, whereas the former are prefer-entially soluble in benzine, the alcohols, ketones, and acids are retained almost entirely in the alcohol layer. In exception to this general classification is cryptoxanthin, which probably because of i t s low oxygen content, is f a i r l y soluble in both phases but goes i f anywhere to the benzine layer. After the above separation has been made, the petrol ether layer may be further subdivided by saponifying which breaks down the esters yielding the free pigments and then redistributing them between the same two solvents. Thus the presence of esters may be detected. The pigment extracts must however be watched for the presence of oils and other interfering substances which may distribute themselves in such a way as to change the solvent properties of one of the phases, and thereby destroy the value of the separation* A considerable number of colour reactions of carotenoids are reported and were widely used by the pioneer investigators. We know now however, that their lack of specificity has been the cause of much trouble, and that though they may be of value when coupled with other evidence, they should not be relied on. We w i l l mention only the reactions with sulphuric acid and with the chloroform solution of antimony trichloride. Concentrated sul-phuric acid i s sometimes used to detect the presence of polyene pigments because in their presence i t produces a blue colour, which seems to depend upon the number of double bonds in sequence, for its shade. Kuhn and Winterstein, working with the compound C5H5-(OH = GE) -Cgttg found that the colour produced depended upon the value of n, thus ~ for n = 1 or 3, no colour; n = 3 , yellow orange; n = 4 , red; n a 5 , violet red; n « 6, blue; n = 7, blue green and n e 8 , blue green. The Oarr-Price colour reaction is the reaction in which a solution of antimony trichloride in chloroform produces a deep blue colour when mixed with vitamin A. It was recommended for the quantitative and -qualitative estimation of this vitamin, but un-fortunately i t has since been found to give the same test with any carotenoid. Another disadvantage of the reaction i s that the colour produced is short lived {about 1 0 - 2 0 second) and spectroscopic ex-amination of the blue colour, which could make the test specific, i therefore very d i f f i c u l t , Various modifications of the test have been proposed but none of them have met with any general approval. The foregoing summary of methods of carotenoid investigation is very brief. If further information is desired the reader would be well advised to read the section on carotenoids in Oilman's "Advanced Organic Chemistry" or some of the papers of Paul Earrer of the chemical properties and methods of investigation of caroten-oids. It is interesting to note that the chromatographic method which today i s deemed so Important was discovered and reported by Tswett in 1906. He used i t in his own investigations, but i t was then forgotten and only recently reinvestigated. It is finding more and more use in the f i e l d of organic chemistry as is shown by i t s recent applications to the separation of sterols and of optically active isomers. The formation in nature of carotenoid pigments is like so many other natural synthesizing processes, not understood. How-ever, several interesting theories have been proposed to account for their formation and i t would not be amiss to consider them here, provided that we keep in mind that none of them have much experi-mental basis. -13-Of these theories, the more logical ones usually consider isoprene (CHg = C(CH5)3-GH = GHg) as the building unit from which , the pigments are built up. The polymerization of isoprene could take place in one of the following three ways: 1* By direct linear head-to-tail union -CJ/^C-CJf-C^i - CAj.-C-OJ"^ ^e/c-+ -C^C-C-C-CJ- C-C - ^./-c . Such addition might go on indefinitely, w i l l form either cyclic or acyclic hydrocarbons and is usually considered to be the origin of the tftrpenes. 3, A similar type of polymerization with the concurrent hydro-genation which would explain the formation of such hydrocarbons as that from which phytol (C20H40°) i s derived.. 3. Addition with subsequent dehydrogenation in the 1:4 pos-ition ^ Jr , ', / w These units then unite to form longer units as in the carotenoids Thus n isoprene units give double bonds and i t is significant to note that the number of such bonds in most of the common carot-enoids is uneven (7, 9, 11 or 13). The Diels - Alder diene synthesis has strengthened these theories by showing the relatively mild conditions under which such reactions can be accomplished, Karrer and his associates early recognized that direct head-.to-tail union of isoprene molecules proceeds to a limited extent and that two of the relatively short chains then unite to a longer one in such a way that one half mirrors the other and a sjjmetrical mole-cule is formed. Considering this fact with polymerization type 3 above we could then postulate the following reactions -c/ = C-'-<ZAg - c-C^=-cJ2 — ^ C-A2 - c-oA*cA-cA* c-cJ^CA^ I If now two of these new molecules were to unite t a i l - t o - t a i l in accordance with Earrer's statement we would have the structure It i s seen that whereas in formula I the methyl groups are in the 1,5 position, in the new compound (formula £1), although the outer methyl groups are in the 1,5 position, the two central ones are with respect to each other, in the 1,6 position. It is rather startling to find that this same arrangment exists in the natural carotenoids as w0ll be seen on examining the formula given on page fr&. Some support is also given to the idea of dehydrogenation in the process by the oxygen requirement of ripen-ing fruit* Karrer and others have advanced another hypothesis to explain -15-carotenoid formation, which is based upon the assumption of -methyl-erotonaldehjide as the primary unit in place of isoprene. yC-cJl-CJo + cJj - £~cA-czJ<o These theories have suggested methods of synthesizing caroten-oids but those tried so far have been unsuccessful. If any of the above theories are to be considered,a problem compound from which the C 4 Q compounds are derived, Willst&tter and Mieg suggested that phytol might play such a role. If such were the case i t would obviously account for the close association of carotenoid and chlorophyll pigments in the green parts of plants since the chlorophyll molecule owes about one third, of its mass to the phytyl group. Furthermore, Karrer and his coworkers have taken completely hydrogenated phytol and then, after converting i t to bfebmide, condensed i t by the action of potassium to a perhydrolycopene of formula O^QHQS which is identical with that obtained from the catal-ytic hydrogenation of the tomata pigment lycopene* In considering phytol as a precursor to carotenoids, the chlorophyll-carotenoid ratio must be examined. It is well known that carotenoid content increases with the break down of chlorophyll and of obvious interest i s the determination of the antecedent Cgo -16-this suggests that the phytol source could be the green pigments themselves. Euhn and Brockman investigated this possibility and reported that the chlorphyll decomposed was insufficient to account for a l l the carotenoid formed. Their results could be taken to suggest that the phytol normally used in chlorophyll synthesis is now being used in carotenoid synthesis and the fact that carotenoid synthesis can proceed in the dark, whereas the green pigments require light for the completion of their synthesis could also be adopted to this idea. A more logical hypothesis, however, seems to be that phytol and the carotenoids have a common ancestor. Such a theory is supported by the presence in organs of plants, of large quantities of carotenoids with no associated chlorophyll. Such organs are the carrot root, and tomatoes which have been grown in the absence of light ( ). Such are the theories which have been propounded to account for the production of carotenoids in plant tissues. It seems probable that the f i r s t product formed would be a hydrocarbon from which could be derived and carotenols and other oxygenated derivatives by direct oxidation. Following is a l i s t of the known pigments of the carotenoid group. The f i r s t l i s t i s that given by P. Karrer (£>) in 1956, the second that of M.T. Bqart (y ) at about the same time. Karrer's l i s t i s somewhat more rigorous i n what is included as a distinct pigment . .  . -17-but most of the pigments included, in Bogart's l i s t are probably distinct: 1. Karrer 1. Anth eraxanth in • 6. Gapsorubin 2, Astacin 7^-Garotene 3. Azafrin 8.^ - Carotene 4. Bixin 9.^-Carotene 5. Capsanthin lOi-Carotene 11.Oryptoxanth in 21.Ehodopurpurin IS.Echinenon 22.E3iodoyibrin 15.Englenarhodon 23, Bhodoviolascin M.llavoibhodin 24.Bhodoxant bin 15. iiavoxanth in 25.Rubixanthin 16.Pucoxanthin 26.Sulcatoxanthin 17.Lycopene 27.T-araxantb.in 18. Pec t enoxantii in 28* Violaxanthin 19*Pentaxanthin 29.Xanthophyll (Lutein' ) SO.Ehodopin 30.Zeasanthin List given by H.Bogart Hame formula Substituents_ Occurence (from place discovered maybe others) Anthers of Lilium tigrinum 1. Anther axanthin C 4 0 H 5 Q O 2. Astacin °4Cf!5803 -18-List given by HyBogart (Cont'd. Name Formula Substituents Occurance S' ^zafrin 027 H38 04 (OK) 2 COOH Escobedia scabrifoli 4. Bixin 5. Gapsanthin 6* Capsorubin 7^-Carotene 8.^- Carotene 9. r-Carotene C25 H30 04 040 H58 03 040 H60 04 C40 H56 C40 H56 040 H56 . (COOH). (C00CH3) Annatto (Bixa orellana) loi-Sarotene C40 H56 11. Citraurin 12. Crocetin C20 H24 04 13.Cryptoxanthin C40 H46 0 14.Cynthiaxanthin (OH)2 0 = 0 (011)2 (C=0)2 (COOH) 2 15. Echinenone CIO H5G ZO° Cu0 ]<>-(,.<..0 16. Eschscholtzxanthin 17. Euglenarhodon 040 H56 03 18. Flavorhodin 19. Flavoxanthin C40 H560& 20. Fucoxanthin C40 1156 06 21. Glycmerin (00)4 Capsicum annum Capsicum annum Carrot - Caucus Carota Carrot - Urtica ureus Carrot - Urtica Ureus Gonocaryum pyriforme Saffron -7.Crocus sativus Ground Clierry-Fhysalis alkakeugi Halocynthia papilosa Sea Urchin Eschscholtzia California^ Euglena h i l -iorubescens Purple Bacteria Ranunculus acer (OH)4 (00)2 Brora Algae • • A -Mussel 22. Isolutein 25. Lycopene C4-0 H56 24. Lycophyll G40 1156 02 (OH) 2 25. Lycoxanthin 040 1156 0 26. Myxoxanthophyll 040 H54-60) 05 27. Liyxoxanthin 040 H54 0 0=0 28. Pectenoxanthin G46 H^ 52-56J 07 29. Pentaxanthin 040 E(54-60j 05 30. Retinene 31. Rhodopin 32. Hhodopurpurin 040 H56-58 -33. Rhodevibrin 34. Rhodoviolascin 042 H60 02 (0CH3)2 35. Ehodoxanthin 040 H50 02 (GO)2 36. Rubixanthin 040 H56 0 OH 37. Salmie acid GOOH 38. Sarcinene Green leaves Lycopersicum esculentum Solanum dulcamara Tomata & Bitter Hight shade berries Myxophyceae do A Mussel Sea Urchin Animal Eyes Purple Bacteria Do Do Do Taxus baccata berries Rosa rubiginosa Salmon Sarcina lutea 39. Spirilloxanthin Sulphur Bacteria 40, Sulcatoxanthin 040 H52 08 ? Sea Anemone • 41. TaraxantLin 040 H56 04 (OH)4? Dandelion 43. Yiolaxanthin 040 1156 04 (OH) 4? Viola tricolor 43, Yiolaerythrin Sea Anemone 44. Xanthophyll s (tutein 040 H56 02 (OH) 2 Green Parts of Plants 45. Zeaxanthin 040 H56 02 (OH) 2 Yellow corn Investigations of recent years have made i t clear that carotene is a provitamin A, and that the vitamin A effect of foods runs essentially parallel with their carotene content. Carotene is transformed i n the animal body into Vitamin A, which like carotene i t s e l f tends to accumulate in the liver. The most obvious explantion of this formation of Vitamin A from carotene i s that i t consists in a simple hydolysis, which cuts the carotene moecule in two, exactly in the middle. e C -c"= CH - t = cn - QH :?/)- e x c H - c /•< e'4 II 0 t//>f^-t .c//-cy=f// - t - e//- o//. ^ c/ This constitution for Vitamin A was proposed and established by Karrer and his coworkers ( f )... A convincing proof of the struct-ure of the carbon skeleton was provided by the synthesis of perhydro-vitamin A from B-ionone and the identity of this synthetic product u with a'perhydrovit amin A obtained by the catalytic reduction of a carefully purified Vitamin A from l i v e r oils. According to the present state of our knowledge of the connection between structure and vitamin A activity, only those compounds which contain the following corr|.ex exhibit provitamin A activity, this complex apparently being easily transformed to Vitamin A in the living organism. CV, CH, Y «*» e/ ^ A change in the location of the double bond In' the ionlne cycle l^-y or the insertion of an OH therein, destroys the Vitamin A effect. Thus t>c & kcarotenes are a l l active, but B-tsomer with two such complexes, is approximately twice as potent as the other two. Sim-i l a r l y , the carotenol cryptoxanthia (3 - Hydroxy - B- carotene) functions as a provitamin A, but rubixanthin ( S - Hydroxy -^6 -carotene) and zeaxanthin (3, 3* - dihydroxy - B- carotene) neither of which contains an unhydroxylated B- ionone, are devoid of such properties. In addition to the three carotenes and cryptoxanthin, the only other naturally occurring carotenoid, and the sole zoocarotenoid reported to show vitamin A activity i s the echinenone of the sea urchin. Although i t s constitution is s t i l l unknown, i t would follow from this property that enchinenone contains the Vitamin A complex. The following synthetic products also show the provitamin A properties: B - Carotene diiodide 040 H56 12 °i - Garolaie Di- Hydro - B - carotene 040 H58 Di-hydro /3 - carotene C40 H58 B - carotene oxide C40 H56 0 Gxy - & - carotene 040 H56 02 Semi - 0 - carotenone 040 H56 02 It is surprising that B - ionone, which is Itself devoid of any provitamin A action, should appear to be so essential a part of those polyenes possessing this property; and that a product herto-fore of interest chiefly because of its violet perfume should suddenly be discovered to be playing a leading role in the field of carotenoids and vitamins. Human vision seems to depend upon the bleaching of the eye's sensitive "visual purple" by the light with the formation of an orange "visual; yellow" who^se color i s said to be due to a yellow pigment related to the carotenoids, termed "retinene" by Wald( CJ ) This retinene i s liberated from visual purple not only by light but also by the action of chloroform. It disappears from the retina Wither by reversion to "visual purple" or by transformation Into vitamin A and other colorless products. In this degredation of visual purple to retinene and Vitamin A and i t s rebuilding from them again, i t is not clear just how much is due to the retinane and how much to the Vitamin A, Some of the latter i s lost in the process, so that the organism requires a constant outside source Z3 • of supply, f o r t h i s automatic r e g u l a t i o n of the eye's sen-s i t i v i t y does not f u n c t i o n p r o p e r l y without i t . At p r e -sent however, th e r e i s no d e f i n i t e evidence t h a t the r e t -inene Is the s o l e , or even the main, source of. Vitamin'A v i n l i g h t adapted r e t i n a s or t h a t i t s s t r u c t u r e i s d i s t i n c t from t h a t o f B - carotene. The use of p i l c h a r d and other f i s h o i l s i n the f e e d -i n g o f c h i c k e n s has r a i s e d s e v e r a l i n t e r e s t i n g problems which are r e l a t e d t o the c a r o t e n o i d pigment content of the o i l s . The pigments of egg y o l k s have been s t u d i e d by H e i l b r o n e t a l (/<?) , Brown ( n ), and o t h e r s . Although the c h a r a c t e r i s t i c pigments are l u t e i n , zeajfanthin and c r y p t o x a n t h i n J^-"T, Brown has shown t h a t the pigment con-t e n t of the d i e t can I n f l u e n c e the p i g m e n t a t i o n of the y o l k to a c o n s i d e r a b l e e x t e n t . A f t e r f e e d i n g c h i c k e n s on a c a r o t e n o i d - f r e e d i e t u n t i l the yolks'-were' f r e e from these pigments, he added pigments p e r i c a r p t o the f e e d as a c a r o t e n o i d source. The s h e l l s c o n t a i n zeaxanthin,, c a p s a n t h i n , c r y p t o x a n t h i n and c a r o t e n e . He added a l s o l a r a x a n t h i n and v i o l a x a n t h l n . The The y o l k s of the eggs were.again a n a l y s e d and the pigments determined. Based upon f e e d consumption and the egg p r o -d u c t i o n , 65$ of the i n g e s t e d x a n t h o p h y l l s are d e p o s i t e d i n , the egg y o l k , but the a b s o r b t i o n of i n d i v i d u a l pigments v a r i e s w i t h i n wide l i m i t s . , L u t e i n and z e a x a n t h i n are d e p o s i t e d t o a much g r e a t e r e x t e n t than c a p s a n t h i n -and c r y p t o x a n t h i n . Lyeapehe and carotenes are a p p a r e n t l y u n a v a i l a b l e as are t a r a x a n t h i h 2k and v i o l a x a n t h i n . I t would appear t h a t the s t r u c t u r e of the pigment has c o n s i d e r a b l e i n f l u e n c e upon i t s d e p o s i t i o n In the y o l k . The hydrocarbons and the h i g h l y oxygenated compounds above show almost no d e p o s i t i o n w h i l e .crypto-x a n t h i n ..containing a s i n g l e h y d r o x y l group i s but l i t t l e b e t t e r . I t seems s i g n i f i g a n t t h a t z e a x a n t h i n and l u t e i n , which are r e s p e c t i v e l y , 3,3- d i h y d r o x y ~f3- carotene and •5,3 - d l h y d r o x y oC - carotene, and which t h e r e f o r e have very s i m i l a r s t r u c t u r e , should be almost e q u a l l y w e l l d e p o s i t e d . A problem c l o s e l y r e l a t e d to the above i s t h a t of the pigment content of the f a t d e p o s i t s of v a r i o u s a n i m a l s . These d e p o s i t s may r e p r e s e n t a p o s s i b l e V i t a m i n - A s t o r a g e , and are t h e r e f o r e o f c o n s i d e r a b l e p h y s i o l o g i c a l i n t e r e s t . Zechmeister and h i s co-workers ( /z ) have g i v e n the probejlrn some a t t e n t i o n i n an attempt t o c l a r i f y the p i c t u r e , but t h e i r o b s e r v a t i o n s have so f a r , served o n l y to complicate the problem. They found t h a t the f a t d e p o s i t s of horse and cow c o n t a i n o n l y c a r o t e n e , whereas those of f o w l s and humans c o n t a i n l u t e i n but no c a r o t e n e . Swine on the other hand, j u d g i n g from the extreme whiteness of the f a t prob-a b l y c a n t a i h no c a r o t e n o i d s . Chalmers has made the i n t e r e s t i n g o b s e r v a t i o n (unpub-l i s h e d ) t h a t c h i c k e n s f e d upon a d i e t .containing p i l c h a r d o i l , s t o r e up i n the l i v e r , a dark brown, pigment. The p o s i t i o n of the l i v e r i n the v i t a m i n - A p i c t u r e suggests a r e l a t i o n between t h i s pigment and members of the c a r o t -e n o i d group, and i t s s t o r a g e i n the l i v e r i s o b v i o u s l y o f in-considerable i n t e r e s t . As a p r e l i m i n a r y i n v e s t i g a t i o n to the study of the problems mentioned above, the determination of the Garot-te enoids of commerca-il p i l c h a r d o i l was undertaken. This o i l i s obtained from the whole f i s h a f t e r a short steam cooking, by pressure. I t i s then " c o l d c l e a r e d " by coo l i n g to remove s t e a r i n s . The o i l thus obtained i s of a dark o l i v e brown c o l o r but may vary condiderably with the season i n which the f i s h are caught. The f a c t that the v i s c e r a of the f i s h are not removed p r i s r to e x t r a c t i o n means that the stomach contents w i l l probably i n f l u e n c e the pigmentation of the o i l , s ince the lower animal forms and algae are very h i g h i n carotenoids, many of them p e c u l i a r to the c l a s s i f i c a t i o n group to which the organism belongs. The most common feed of p i l c h a r d s i s sa i d to be the "red feed" which i s composed of small crustaceae, the cocopods. Since i n the lower animals forms the most common pigment i s a s t a c i n , i t seems probable that the cocopods might introduce t h i s caretfioid i n t o the o i l . However, i f the feed contained diatoms or other algae, i t might be expected that numerous other pigments such as fucoxanthin, c h a r a c t e r i s t i c of the brown algae and myxo-xanthin cemyxoxanthophyll c h a r a c t e r i s t i c of the blue green, algae, might be present. Both of these groups contain small forms and so could conceivably form p a r t of the feed. E x p e r i m e n t a l Procedure. The s e p a r a t i o n of the pigments o f p i l c h a r d o i l p r e -sented a problem of unusual i n t e r e s t . Examinations of t h i s o i l have been made by Tompkins and by B a i l e y {/U) . Tompkins r e p o r t e d the presence i n the o i l of c h l o r o p h y l l which may g i v e a d i s t i n c t g r e e n i s h t i n t and which together w i t h the r e d and y e l l o w pigments undoubtedly accounts f o r the u s u a l o l i v e brown c o l o r . B a i l e y examined the c a r o t e n o i d content of p i l c h a r d o i l and r e p o r t e d the presence of carotene, x a n t h o p h y l l and f u c o x a n t h i n , He used s m a l l samples of o i l ( 5 0 c c ) , d i s -s o l v e d i n C S ^ , which he passed over a column of alumina. The pigments o b t a i n e d were i d e n t i f i e d by t h e i r s o l u b i l i t y , and c o l o r r e a c t i o n s # p e i t i o n s on the column. B a i l e y a l s o ex-amined canned p i l c h a r d s i e . f i s h from which the v i s c e r a has. been removed and found t h a t f u c o x a n t h i n was absent from the o i l . When sommercial o i l was t r e a t e d i n a manner s i m i l a r to the cann i n g p r o c e s s , f u c o x a n t h i n was not d i s t r o y e d . B a i l e y t h e r e f o r e concluded t h a t the source o f the f u c o x -a n t h i n was the v i s c e r a . He g i v e s the f o l l o w i n g pigment con-c e n t r a t i o n s f o r a dark c o l o r e d o i l : C a r o t e n e — Q.Ot - 0. 2 - i ' p ^ r \oocf- oli X a n t h o p h y l l — °^ U h U] Fuc o x a n t h i n - - u I t i s w e l l known from the work of H e i l b r o n and K a r r e r [d) that f u c o x a n t h i n p r e s e n t i n f i s h brown algae goes over t o z e a x a n t h i n i n d r y i n g of the a l g a . Hi|elbron ( ) has r e p o r t e d t h a t t h i s same change takes p l a c e on the a d s o r b t i o n 27 column, while Zechmeister ( ) has given evidence that i t takes place i n s o l u t i o n . I t seems po s s i b l e therefore that Zeaxanthin may a l s o be present i n p i l c h a r d o i l , although B a i l e y f a i l e d to observe any sign of i t . Zechmeister (^) i n h i s examination of the pigments of horse and hen f a t reported an attempt to separate the p i g -ments by shaking up the melted f a t with the adsorbent. We have t r i e d to apply t h i s method to the p i l c h a r d o i l , but ob-tained poor separation. I t i s usual i n removing o i l from carotenoid e x t r a c t s to saponify the mixture with a l c o h o l i c KOH, and then to ex-t r a c t the mixture with ether or p e t r o l ether, a f t e r the ad-d i t i o n of water. This method i s s a t i s f a c t o r y when the qu a n t i t y of o i l i s small,but In t h i s case presented con-s i d e r a b l e d i f f i c u l t i e s . F i r s t , the amount of soap formed was extremely lar g e and p r e c i p i t a t e d upon a d d i t i o n of only small amounts of water. This p r e c i p i t a t i o n i n t e r f e r e d s e r i o u s l y with any attempts at e x t r a c t i o n of the pigments from the s a p o n i f i e d mixture probably p a r t l y due to adsorb-t i o n of pigment upon the soap but more because i t tended to destroy the i n t e r f a c e thus preventing separation. I f jus t s u f f i c i e n t water was added not to cause p r e c i p i t a t i o n a b e t t e r separation was obtained but large amounts of the e x t r a c t i n g solvent d i s s o l v e d i n the a l c o h o l l a y e r . F u r ther-more c e r t a i n of the pigments were found to be exceedingly s o l u b l e i n a l c o h o l and therefore tended to remain i n the a l c o h o l i c l a y e r . An attempt was made to remove the soaps by s a l t i n g out or by p r e c i p i t a t i o n as c a l c i u m s a l t s of the a c i d s . 'The removal and washing of the p r e c i p i t a t e s how-ever was very d i f f i c u l t and because of the f e a r ,of l o s i n g pigments i n the p r e c i p i t a t e ^ e i t h e r through a d s o r b t i o n ^ o r . f a i l u r e to'.wash t h o r o u g h l y , t h i s method was abandoned. The p o s s i b i l i t y of e x t r a c t i n g the pigments from the o i l was then examined. F a t s s o l v e n t s were of course of no use. <0f the o t h e r s , a l c o h o l s were the o n l y ones which showed any promise,?methanol was l e a s t s o l u b l e i n the o i l . By comparison w i t h a chromatograph, the e f f e c t of an a l c o h o l e x t r a c t i o n was determined. The e x t r a c t i o n was made by e m u l s i f y i n g o i l and a l c o h o l i n the r a t i o of two volumes of o i l t o one of methanol, a t 50 degrees. The emulsion was a l l o w e d t o break and the l a y e r s s e p a r a t e d . Three such ex-t r a c t i o n s s erved t o remove by f a r the g r e a t e r p a r t of the e x t r a c t a b l e pigment, but the chromatogram showed t h a t o n l y one of the pigments was removed. The a l c o h o l e x t r a c t e d was evaporated down to a dark o i l y r e s i d u e ) w h i c h was d i s s o l v e d i n et h e r and p e t r o l e t h e r * On'cooling;some amorphous'/white m a t e r i a l p r e c i p i t a t e d and was f i l t e r e d o f f . The chromatograph of t h i s e x t r a c t showed pr e d o m i n a n t l y one orange-red band which was l a t e r i d e n t i f i e d f u c o x a n t h i n ' a n d t r a c e s of one or two other y e l l o w bands and some green m a t e r i a l . S i n c e the y e l l o w pigments were p r e s e n t i n t r a c e s o n l y , i t was concluded t h a t they were p r o b a b l y c a r r i e d over m e c h a n i c a l l y i n o i l drops or were d i s t r i b u t e d v e r y s l i g h t l y i n the a l c o h o l . B e s i d e s f u c o x a n t h i n , the e x t r a c t c o n t a i n s l a r g e amounts of s t e r o l l i k e m a t e r i a l together w i t h some o i l , p r o b a b l y r a t h e r h i g h l y u n s a t u r a t e d ^ j u d g i n g from the i n t e n s i t y of the r e d c o l o r produced upon s a p o n i f i c a t i o n . These waxy . m a t e r i a l s are r e a d i l y adsorbed on the chrom atograph and have the e f f e c t of cementing tog e t h e r the adsorbent m a t e r i a l t o make i t almost impermeable t o the pigment s o l u t i o n . I t was thought t h a t perhp\as the a d d i t i o n of p e t r o l e t h e r t o the o i l might reduce the s o l u b i l i t y of the x a n t h o p h y l l f r a c t i o n s u f f i c i e n t l y t o a l l o w e x t r a c t i o n w i t h a l c o h o l . I t was un-s u c c e s s f u l however, because the presence of s m a l l amounts of o i l i n benzine makes i t a f a i r l y good s o l v e n t f o r c a r o t -enoids. Another d i f f i c u l t y was caused by the change i n s p e c i f i c g r a v i t i e s brought about. Methanol i s i n t e r m e d i a t e i n s o l u b i l i t y between o i l and p e t r o l e t h e r but a mixture of o i l and p e t r o l e t h e r has a s p e c i f i c g r a v i t y v e r y c l o s e to t h a t of the a l c o h o l and s e p a r a t i o n i s v e r y slow. The method f i n a l l y adopted f o r s e p a r a t i n g the pigments was the chroiuatographing of the o i l i t s e l f . P r e l i m i n a r y experiments i n d i c a t e d that a c t i v a t e d alumina was more s a t -i s f a c t o r y than a c t i v a t e d magnesia f o r the purpose. Mag-n e s i a i s not s u f f i c i e n t l y s t r o n g and a l l o w s the pigments to pass through the column too r a p i d l y but has the f u r t h e r d i s -advantage of becoming a d i r t y brown c o l o r i n the presence of o i l which tends t o s c r e e n the l i g h t e r bands. I n e a r l y ex-p e r i m e n t s the o i l was d i s s o l v e d i n l i g h t p e t r o l e u m t o g i v e more r a p i d f i l t r a t i o n but i t was l a t e r found t h a t the pure o i l was e q u a l l y s a t i s f a c t o r y and was t h e r e a f t e r used. Glass tubes f i v e f e e t long and f i v e cm. i n diameter were packed to a height of 45 cm. with a mixture of act-i v a t e d alumina ( A l o r c o — 1 0 0 mesh) and a f i l t e r - a i d (Johns-M a n v i l l e H y f l o Super-cel) i n equal parts by weight. The ;, c a p acity of such a column was about one l i t r e of o i l . When that volume had been passed through, the lower bands had reached the bottom of the column and washing was u s u a l l y impossible. Three columns were set up and the o i l passed over them. The f o l l o w i n g zones developed: (1) A broad green band cov-e r i n g the top four or f i v e cms. of the column. (2) Three bands close together, the upper two orange red and the t h i r d one orange. (3) Considerably below these, two bands close together, the upper one green, the lower one yellow. The f i l t r a t e from the columns was l i g h t yellow. The lower yellow and green bands were allowed to pass through the column i n order to o b t a i n better s e p a r a t i o n of the upper bands. The adsorbent was pushed out and the columns, s t i l l con-t a i n i n g considerable q u a n t i t i e s of o i l , were d i v i d e d i n t o three p a r t s corresponding to the green upper zone, the c e n t r a l zone c o n t a i n i n g the three pigments, and a lower zone c o n t a i n i n g no c o l o r i n g matter. The green pigment at the extreme top was very t i g h t l y adsorbed. An alcomol e x t r a c t of the o i l removes some of the green but on shaking the a l c o h o l e x t r a c t with p e t r o l ether t h i s pigment goes over i n t o the upper l a y e r . T h e , p o s i t i o n of the bands on the column, together with the s o l u b i l i t y and 3* a b s o r b t i o n maximum. (686 >^ 610 -yx» and 537->^co i n o i l ) i n -d i c a t e t h a t the green m a t e r i a l s are c h l o r o p h y l l s and der-i v a t i v e s . The adsorbed c a r o t e n o i d s of the c e n t r a l zone were e l u t e d w i t h methanol w i t h o u t any attempt t o separate them, and were then t r a n s f e r r e d to a mixture of c h l o r o f o r m and carbon d i s u l p h i d e and a f t e r c o n c e n t r a t i n g and d r y i n g over sodium s u l p h a t e , were reghromatographed on alumina. Two deep y e l l o w and one l i g h t y e l l o w band appeared. Of these, one of the deep y e l l o w bands remained t i g h t l y adsorbed upon the column but the other two passed through i n t o the f i l -t r a t e upon washing w i t h more of the s o l v e n t . The adsorbed pigment was e l u t e d w i t h methanol and then cromatagraphed from CS 2 ^ and washed w e l l w i t h 570 a l c o h o l i c p e t r o l e t h e r . A f t e r e l u t i o n , i t was evaporated twi c e to dryness from CS 2 , redis s c b l v e d i n 1-2 cc. of CS and a l l o w e d t o s t a n d o v e r n i g h t i n the i c e box. A f t e r f i l t e r i n g , the s o l u t i o n , p e t r o l - e t h e r (B.P. 35-60) was added and the m i x t u r e c o o l e d . An e s t i m a t e d one mg. of pigment was ob-t a i n e d and r e d i s s o l v e d i n CS^. The amount of p e t r o l ether added f o r c r y s t a l l i z a t i o n appears important because i f i t i s not c o r r e c t a r e s i n o u s p r e c i p i t a t e i s o b t a i n e d . T h i s pigment showed a b s o r b t i o n maxima of 506, 476, 444 iry* i n CS^ ; remained c o m p l e t e l y i n the a l c o h o l l a y e r upon d i s t r i b u t i o n between p e t r o l e t h e r and 70fo a l c o h o l ; and gave a green c o l o r w i t h s u l p h u r i c a c i d . I t was concluded to be f u c o x a n t h i n . The f i l t r a t e from the column on which the f u c o x a n t h i n was adsorbed, was evaporated almost to dryness and r e d i s -s o l v e d i n p e t r o l e t h e r , and chromatographed on a column of alumina. A deep y e l l o w band remained on the column and the f i l t r a t e came through deep y e l l o w . The o r i g i n a l o i l f i l t r a t e c o n t a i n i n g the lower green and y e l l o w bands w i t h some of the l i g h t y e l l o w o i l which name through f i r s t was then s a p o n i f i e d and e x t r a c t e d w i t h p e t r o l e t h e r a f t e r c a r e f u l a d d i t i o n of water. This s o l u t i o r was c o n c e n t r a t e d and adsorbed on a column of two p a r t s mag-n e s i a and one p a r t of f i l t e r a i d . A d i f f u s e y e l l o w band former on the column and was e l u t e d w i t h methanol. The f i l t r a t e from the column, which was l i g h t y e l l o w i n c o l o r , was not adsorbed upon alumina or magnesia from p e t r o l e t h e r , was s o l u b l e i n p e t r o l e t h e r and not i n a l c o h o l , and so c o r -responded to the pigment of the o i l f i l t r a t e f i r s t o b t a i n e d . T h i s pigment which i s p r o b a b l y a hydrocarbon, must be the carotene of B a i l e y . I t s c o n c e n t r a t i o n i n the o r i g i n a l f i l t r a t e , as determined by ¥. Chalmers w i t h the t i n t o m e t e r , corresponds to a p p r o x i m a t e l y .06 mgs. carotene per 100 cc. of o i l . BIBLIOGRAPHY. 1. Cook Chemistry and Industry 2. H e i l b r o n and E l R i d i . Biochem..J 3. H e i l b r o n and E l R i d i i b i d , 4. S t r a i n Jour*. Bi©l, Chom. Z, P h y s i o l . Chem Helv. .Chim.. Acta. "Organic Chemistry' •H-elv* Chim. Acta Science 10. G i l l a m and H e i l b r o n Biochem. J 11, Brown Jour. B i o l . Chem* 5. K y i i n 6, KKarr'er 7:.; Gilraan 8. 'Karrer 0. Wald %bt 7 24, (1936$) 30, 1735 (1936) 31, 1605 (1937) 105, 523, (1934) 166, 39, (1927) 19, E33, (1936) John Wiley & Sons, 1938 14, 1036, (1931) 82 No.2136 (1935) 29, 1064, (1935) 122, 625, (1937-38) 1 2 . Zechmeister and.Tuscon Z. P h y s i o l . Chem" 231, 259, (19' 13. Tompkins O i l and Pat industr y 7, 5 5 , ( 1 9 3 0 ) 14. ; B a i l e y Jour. F i s h Res. Bd. Can. 4. 1, (1938) 15. H e i l b r o n et a l , Biochem. Jour. 29, 1369, (T935) 16. K a r r e r , Rttbel, and Strong Hely. Chim. Acta. 19, 29, (1 


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