Metabolic fractionation, storage and display of carotenoid pigments by flamingoes

Comp. Biochem. Physiol., 1962, Vol. 6, pp. 1 to 40. Pergamon Press Ltd., London. Printed in Great Britain

METABOLIC FRACTIONATION, STORAGE AND DISPLAY OF CAROTENOID PIGMENTS BY FLAMINGOES D, L. FOX Department of Marine Biology, Scripps Institution of Oceanography, University of California (Received 22 ffanuary 1962)

A b s t r a c t - - 1 . Captive specimens of the brilliantly coloured American or West Indian flamingo, Phoenicopterus ruber, and its paler South American relative, Phoenicopterus chilemis, gradually lose the red pigmentation of the exposed skin, and of the feathers through moulting, unless fed a diet rich in carotenoids. 2. Analyses of the brightly coloured feathers, exposed leg-skin, egg-yolk, blood plasma and various internal organs from captive flocks have revealed rich stores of carotenoids, including prominently eanthaxanthin and varying amounts of astaxanthin in some sites, and numerous other apparently oxidized members not yet completely characterized. 3. Astaxanthin, present in adult feathers of P. tuber and in the exposed leg-skin of both species (as an ester in P. chilensis), was not detected in the plasma or in any internal organs or tissues examined, save for suspected traces in but one of several blood-filled shafts of young pin-feathers examined chemically directly after plucking from living adult P. tuber. T h e small, rare species, Phoenicoparrusjamesi, deposits both canthaxanthin and astaxanthin in its feathers. 4. The pale and always largely fluid faeces, taken from the intestines of freshly dead adult P. ruber and P. chilensis specimens, yielded no carotenoids, or at times only bare traces of yellow pigment, to extracting solvents. 5. Aerobic and anaerobic bacteria from P. ruber's gut exhibited dehydrogenase activity on numerous pure substrates, and altered astacene chemically. 6. Representative concentrations of total carotenoids, in mg per 100 g dry weight, were found to be approximately as follows, in four prominent depots: Tarsal skin 270: Feathers 32: Blood plasma 5: Yolk 4. 7. Newly hatched, white or greyish-downed chicks were without detectable carotenoids in any tissue examined. Their short, oedematous legs show bright pink to red colours from the presence of haemoglobin beneath the thin, naked skin. However, in about 8 days, as a chick prepares to leave its nest, wherein it earlier received intermittent shading by a hovering parent or by sitting on its legs, to migrate into regions of exposure to full sunlight, this skin turns black with melanin deposits. Red carotenoids are later deposited in new feathers and in the naked leg-skin, gradually replacing melanin in the latter as the young bird grows toward adulthood. INTRODUCTION AMONG the v e r t e b r a t e s , b i r d s rival even t h e fishes in t h e i r s t o r a g e a n d e x t r a v a g a n t d i s p l a y o f red, o r a n g e a n d y e l l o w l i p o c h r o m i c o r c a r o t e n o i d p i g m e n t s d e r i v e d o r i g i n a l l y f r o m t h e food. M o r e o v e r , like s o m e fishes, v a r i o u s avian species m o d i f y

2

D.L. Fox

certain dietary carotenoids and subsequently store and display the resulting coloured products (Brockmann & V61ker, 1934; V61ker, 1950, 1954a, b, 1955a, b, 1958; Fox, 1953, 1962; Conway, 1958, 1959; Poulsen, 1960). Among the most interesting birds in this respect are flamingoes, notably the American or West Indian species Phoenicopterus ruber, the largest brilliantly coloured birds of the New World. They display, under natural or certain experimental nutritional conditions, striking pink to vermilion carotenoid pigmentation in the naked skin of their webbed toes, tarsals, heel-joints, tibiae and lower bill-mandibles, as well as in the feathers (Fox, 1955; V61ker, 1958; Conway, 1958, 1959; Poulsen, 1960). There is but minor if any sexual dichromatism in this species, unless mature males may have more highly pigmented feathers, and no sexual dimorphism sa~e that fully adult males attain larger size (Allen, 1956). It is well recognized that captive flamingoes gradually lose the rich pigmentation of their exposed skin, and that new plumage appears successively paler after each moult, finally emerging as white feathers, unless appropriate sources of continuous pigmentary renewal are supplied in the diet (Fox, Conway, Poulsen, op. cit.). It is not unlikely, however, that captive flamingoes, receiving carotenoid-rich food, may as a result assume a coloration departing somewhat from the delicate rosered hues observed in some wild specimens, since the latter have access to a widely variable and incompletely known diet. In view of the challenging biochemical problems posed by these birds, it will be of interest to survey briefly what is known of their habitats and food. Flamingoes inhabit watery inland or coastal areas in tropical or subtropical regions around the world. They display physiological adaptation to widely heterosmotic conditions, ingesting their food from waters differing in salinity from nearly zero in fresh mountain lakes or inland swamps, through brackish concentrations at rivermouths, to the fully saturated salt-encrusted, muddy ooze bordering desert lakes or lagoons (Gifford, 1913; McCann, 1939; Gallet, 1950; Ridley, 1954; Allen, 1956). Moreover, some flamingoes tolerate alkaline waters, e.g. in inland lakes at altitudes of from 3000 to 6000 ft, near the equator in Kenya, where alkalinities, derived from laval sodium salts, may attain concentrations ~f 0.II, ().22 or 0.27 N, giving pit values of from 9-0 to 11-2 (Jenkin, 1929). We are informed by McCann (1939) that the shallow, salt-concentrated, mudladen waters of flamingo habitats in the Great Rann of Cutch on the margin of the Great Indian Desert may reach temperatures of from 64: to 84' in January, rising through 90 ° and even exceeding 100 ~ 1- in July. There, and in other such sites, flamingoes breed, build their conical mud-nests and raise their young. Gallet (1950) states that, of all birds observed, flamingoes tolerate the greatest span of hot and cold conditions, the highest salinities and the most severe paucity of fl~od. Hillaby (1956) suggests, h~wever, that a sudden cold snap during February of 19.% may have contributed to the death of many members of a Camargue flamingo population. Their highly specialized filtering equipment enables flamingoes to utilize a diet of small aquatic plants and animals, seeds and organic detritus, e.g. suspended

METABOLIC FRACTIONATION, STORAGE AND DISPLAY OF CAROTENOID pIGMENTS

3

and sedimentary mud (Jenkin, 1957). There is a strong likelihood that the large size and unique structure of the tongue and the relatively small aperture of the throat would preclude the flamingoes swallowing food items much larger than small cyprinodont fishes (Allen, 1956). Gallet (1950) emphasizes the birds' ingestion of watery mud in great quantities, and believes that this kind of material, notably when rich in organic matter (6--8 per cent), may constitUte a principal source of their nutrition at certain sites and seasons. Allen (1956) points out that the organic content of the gelatinous sapropelic layer on top of the sedimentary mud of the salina at Inagua is very high, some samples exceeding 90 per cent. Thus the American flamingoes feeding in these waters are assured of a diet rich in detrital material, i.e. "organic ooze" or slime inhabited by countless small organisms. In addition to quantities of dark, foul mud and sand, other materials consumed by flamingoes include rotifers, corixids, chironomid and other insect larvae, small crustaceans including copepods and the brine shrimp Artemia salina, molluscs, notably the marine snail Cerithium, fish spawn, diatoms in large numbers, much blue-green algal material of many species, and fragments of higher plants as well as seeds, notably of Ruppia rosellata and Scirpus maritima (Gifford, 1913; Jenkin, 1929, 1957; MeCann, 1939; Ali, 1945; Gallet, 1950; Ridley, 1954; Ridley, Moss & Percy, 1955 ; Allen, 1956). Allen lists species of red sulphur bacteria and other mud-living micro-organisms, as well ~s forms of blue-green algae, diatoms and protozoa which, in the company of certain gastropod and pelecypod molluscs, small arthropods and nemathelminth worms, inhabit the rich muds of certain lakes and salines in the West Indian habitats of Phoenicopterus ruber. The brine shrimp Artemia salina, which stores in its eggs and nauplii considerable amounts of esterified astaxanthin, accompanied by a suspected ketocarotenoid precursor thereto, among other earotenoids (Gilchrist & Green, 1960), occurs widely in saline water-bodies, and probably constitutes a part of the natural food of flamingoes. Allen discusses also the habitats and food of the half-dozen flamingo species known throughout the world, pointing out that their general niche "in the composite form . . . is a shallow lake, its waters brackish to heavily saline, more often the latter. It is isolated, desolate of aspect and lies in a remote corner of a sterile and desert-like region inhabited by few (other) animals." He refers to the wide variety of animal, plant, bacterial and sapropelic food consumed and emphasizes the significant finding that most of the plants eaten have high salinity tolerance, thus reflecting the birds' customary type of habitat. A few previous observations have been made on tissues or feathers of flamingoes. Manunta (1939) extracted from the yellow fat of a flamingo (species not given) 70 g of a yellow-orange oil which, following saponification in methanolic potash under a nitrogen atmosphere, yielded rose-violet crystals. These exhibited in pyridine a single absorption band with a maximum centred at 487 m/z (not at 490500 m/z as shown by astaxanthin or astacene in this solvent). The new compound differed from astacene not only somewhat in its absorption maximum but in

4

D.L. Fox

solubility in, and colours imparted to, various organic solvents, as well as by its chromatographic separability from the reference compound. Likewise V61ker (1954b) reported the recovery, from the feathers of Phoenicopterus ruber, of red carotenoid material which, while reminiscent of astaxanthin in absorption maximum, failed to exhibit the acidogenic characteristics of that compound, i.e. the genesis of astacene upon treatment with alcoholic alkali (in air). It was, moreover, chromatographically separable from astacene itself. In all probability this was canthaxanthin, identified in the present studies of feathers from the same species (Fox, 1960) and from the scarlet ibis (Fox, 1962). Astaxanthin has been reported in several other bird species by various authors. Brockmann & V/~lker (1934) found it in the yolk of eggs from both the stork, Ciconia ciconia, and the laughing gull, Larus ridibundus; these authors, as well as Kuhn, Stene & S6rensen (1939), found the same carotenoid in the red facial skin of the pheasant, Phasianus colchicus, and V61ker (1950) extracted it from the feathers of the shrike, Laniarius atrococcineus. Wald & Zussman (1937, 1938) reported it also in the retina of the domestic hen, and, while they detected none in the yolk, reported it in the retina of the unhatched chick. Astaxanthin may occur more widely among birds than has been suspected. Personal research affiliation with the San Diego Zoo has provided opportunities to observe initially the gradual fading of skin and feather pigments in the flamingo collections; to have the valuable co:operation of Zoo personnel in an extended feeding programme designed to restore the pigmentation; and finally, during the feeding r6gime, to acquire for biochemical studies tissues of any freshly expired specimens, and to have feathers, blood and egg material from living birds as well. EXPERIMENq'AI. Feeding and care The present researches have substantiated the reported recovery, inter alia, of neutral xanthophyIls with single absorption maxima from feathers, skin, blood, and other flamingo tissues. But the work has also revealed the recoverability of astaxanthin and canthaxanthin from pyridine extracts of feathers, and of astaxanthin, convertible to astacene, from extracts of the leg-skin of birds maintained upon a diet containing astaxanthin (Fox, 1955), as well as from feathers of flamingoes maintained on astaxanthin-free diets, rich in other carotenoids. The investigation was pursued primarily on a captive flock receiving a supervised diet containing materials designed to restore or maintain the birds' pigmentation. The San Diego flock is maintained in a spacious enclosure containing a large, fairly shallow concrete wading lagoon supplied with running fresh water, a small concrete feeding pool, an extensive, sunlit lawn and an area shaded by trees. A powerful fountain spills water into the wading pool. During these studies, a dozen additional flamingoes shared, with six roseate spoonbills and a like number of boobies, a more restricted but similarly equipped enclosure in the Zoo's bird canyon. Twice daily, at about 8.30 a.m. and 4.30 p.m., crocks of finely

MBTABOLIC FRACTIONATION, STORAGE AND DISPLAY OF CAROTENOID PIGMENTS

5

comminuted food arc placed in the feeding pools. These pools are cleaned daily, and the wading pools less frequently. The daily diet provided per bird at each feeding was as follows, thus offering approximately 2 lb of food (wet basis) per day to each individual: Cooked brown rice Cooked whole yellow millet Cooked whole wheat Raw ground carrots "Kibble" dog food* White bread Fresh ground horse liver Fresh ground shrimp Fresh (or canned) ground red salmon Fresh shredded lettuce Ground (cooked) crayfish shell Vitamyein (a vitamin-mineral supplement)

1 oz 1 oz 1 oz 1 oz 2 oz 1 oz 1 oz 2 oz 2 oz 1 oz 2 oz 2g

Needless to add, we have by no means replicated natural conditions in the maintenance of these captive birds. The mild climate of San Diego is in great contrast with the rigorous extremes of temperature, notably in the high ranges, endured by wild flamingoes. Although we are at present unable to surmise the variety, quantities and ratios of animal and plant proteins and other components comprising the seasonal diets of the free birds, there is some ground for supposing that our captives receive far more food each day than may be collected by their wild kin. Finally, the San Diego flock had, during the course of these investigations, received only fresh water, rather than the saline kind which characterizes their usual natural habitat. This factor, which may be important in the total economy of the species, is subject to some ready adjustment by supplying access to natural brine in special containers. This was done. Concentrated whole brine was obtained through the generosity of the Western Salt Company of Chula Vista. Several gallons (5-10) were made available in a special large rubber trough near the feeding pool, and the birds have been seen occasionally sampling the brine. Since a large American flamingo weighs some 6-7 lb on the average (Gallet, 1950; Allen, 1956; K. C. Lint, personal communication), those in our San Diego flock receive nearly one-third of their body-weight of food per day (or perhaps closer to one-quarter, allowing for wastage). * Hy-V. O. Kibble Dog Food (Clover Laboratories, Glendale, California) contains a great variety of plant and animal meal, oils, vitamins and salts. Its guaranteed analysis r e a d s as f o l l o w s :

Protein Fat Fibre

not less than 21% not less than 3% not less than 3%

N-free extract Ash Moisture

not less than 45% not more than 8% not more than 9%

6

D.L. Fox

When, in March, 1954, it was apparent that the flamingoes were losing their pigment on the diet then given them, finely ground carapaces of the "California spiny lobster" (actually the large marine crayfish Panulirus interruptus) were added to the food. The crustacean shell, provided in previously cooked condition by local restaurants, is rendered brittle by quick freezing, is next passed through a food-mincer, and finally ground in a blender. Dried shrimp and dried flies, formerly a part of the birds' diet, were no longer included since they contributed little or no carotenoids. Within a few months members of the original flock of twenty American flamingoes (Phoenicopterus tuber) exhibited a conspicuous return of pink to salmon or vermilion colours in both the naked leg-skin, as previously described, and in the new feathers. Members of the smaller, paler Chilean species (Phoenicopterus chilensis) likewise developed bright pink to red colours in the skin of the heel-joint and feet, and gradually also in some of their long wing- and tail-feathers. The further supplement of red salmon flesh, begun in November, 1955, provided the same prominent carotenoid, astaxanthin, as is present in the crustacean carapace, but in augmented quantities and doubtless in more readily available form, being dispersed in protein and lipids of the flesh rather than in the tough, chitinous medium of the crayfish skeleton. This salmon supplement augmented considerably further the visible pigmentation in both species. Both the American and Chilean birds exhibit brightly coloured flight feathers (the former far more so), and the pink-orange neck-feathers of P. tuber are in some contrast with the formerly snowy-white (now delicate pink) necks of P. chilensis. The plumage and skin colours of the younger flock of P. ruber in the canyon became equally arresting, and the young spoonbills (Ajaia ajaja), originally white, now exhibit both soft and quite pronounced shades of pink or rose colours throughout their feathers. Materials analysed for carotenoids were: feathers from living or freshly dead specimens, blood from living adults, skin, internal organs and faeces from freshly dead adult birds, the bright orange yolk of a fresh egg, viscera and black heel-skin from a 16-day-old chick, and pink tarsal skin from a 4-day-old chick. Since these valuable birds arc extraordinarily timid and, when pursued or restrained, may die from shock and internal haemorrhages, or from accidentally broken legs (Gallet, 1950; and observations at the San Diego Zoo), no programme was undertaken for following changes in concentration of blood carotenoids. Blood was obtained from several live birds on two occasions, however; once from wingveins while trimming flight feathers, and later from the shafts of pin-feathers drawn while pinioning the birds to preclude their escape by flight. Fresh skin and other tissues were available only from individuals which had died from some cause.

Chemical procedures Operations involved (1) extraction of the carotenoids, (2) their initial partitional resolution into (a) epiphasic components, i.e. preferentially soluble in a petroleum ether phase, and (b) hypophasic fractions, migrating instead into an

METABOLIC FRACTIONATION, STORAGE AND DISPLAY OF CAROTENOID PIGMENTS

7

underlayer of slightly aqueous methanol (usually diluted by 5 per cent or 10 per cent water, v/v), and (3) the further separation of individual components in each fraction, whether before or following saponification treatment with ethanolic alkali, by powder-column chromatography of their petroleum ether solutions. All of these steps were conducted in general accordance with the outline described and tabulated by Fox & Pantin (1941) and Fox (1953). A few substitutions were employed, however; ethanol was usually employed as an extracting agent in place of acetone, since the latter, while excluding phospholipids, tends, when diluted and shaken with petroleum ether, to give rise to troublesome emulsions; moreover, in contrast to ethanol, acetone is more difficult to wash out of petroleum ether solution. In view of the lability of carotenoids, time-consuming steps were avoided whenever possible. Sodium hydroxide was preferable to potassium hydroxide since it yielded an astacene salt more readily recoverable from aqueous-petroleum ether interfaces. Finally, petroleum ether for the neutral carotenoids, and pyridine as well for the acidic derivative astacene, were usually employed as reference solvents instead of carbon disulphide for spectrophotometric studies. Intimate mixtures of calcium carbonate and celite (2:1), or more often of magnesium oxide and celite (1:1 or 2:1), all of extreme fineness, were packed under suction with a light tamp into Zechmeister-Cholnoky chromatographic tubes, each bearing a thin pad of absorbent cotton upon the sintered glass filterdisk at the bottom. First pure petroleum ether, to saturate the powder, then the same containing the extracted carotenoids, was slowly drawn through the column with a Fisher Filtrator. Developing solvents were added to the petroleum ether to resolve chromatographic zones. Illuminating gas* was frequently admitted, by a glass tube passing through a perforated cork, into the system in place of allowing air to replace fluid, as the solvent was drawn down through the column. Finally, after pressing the caked powder column from the tube with a tamp applied steadily against the cotton pad at the lower end, each coloured zone of adsorbed pigment fraction was removed separately by whittling it away with a spatula into a small receiving beaker; or alternatively, each coloured adsorption band was dug carefully out of the column in situ with a narrow spatula, and similarly segregated. The adsorbed pigment of each chromatographic fraction was then eluted from the powder with petroleum ether now containing traces of methanol, ethanol or benzene. In the latter instance, the whole filtered solution was evaporated in a stream of illuminating gas, and the pigment residue redissolved in petroleum ether. Alcohol, when used as the eluant, was readily washed away with a few successive rinses of distilled water; the remaining petroleum ether solution of pigments was then, if necessary, dried to clarity with solid sodium chloride, and the final solution was examined spectroscopically, often at first with a Hartridge Reversion Spectroscope. A Beckman Model DU photoelectric spectrophotometer, and, in latter * Whose sulphur content is reportedly of the order of only 2 parts per billion.

8

D.L. Fox

phases of the work on feather-carotenoids, a Carey Recording Photoelectric Spectrophotometer (Model 14) was used for determination of the absorption curves. Petroleum ether of boiling range ca. 30-60°C was used throughout the work. Mixed chromatography, for confirming the identity of a carotenoid fraction (e.g. astaxanthin, astacene and canthaxanthin), involved the usual triple operation, wherein one powder column receives the pigment fraction under test, a second the known reference pigment, and the third a mixture of these two; positive identification is given by the identical appearance of all three developed chromatograms, in particular by the presence of but a single coloured zone of the critical component in the third tube, containing the mixture. Saponification was carried out by exposing the pigment to alcoholic potassium or sodium hydroxide (ca. 2-5 per cent w/v) in a warm water bath for a half-hour to an hour and a half, or by allowing the stoppered system to stand in the dark at room temperatures overnight. In dealing with certain fatty materials (e.g. egg-yolk) it was possible to separate at least the readily solidifiable (saturated) lipid components by (a) primary extraction with acetone to exclude phospholipids and (b) chilling to ca. - 2 0 ° C the relatively concentrated petroleum ether solutions of chromatographed pigment fractions, resulting in the precipitation of white lipid materials, which could bc removed by filtering the chilled system. The following lettered designations, or various combinations thereof, are used in the text and with some of the charted absorption curves: E = an epiphasic fraction (preferentially soluble in petroleum ether). H--a

hypophasic component (migrating methanol phase).

preferentially to the aqueous

I -- an interfacial salt, gathering at the junction of the two phases. h -- hydrolysed, or subjected to alkaline saponification treatment. Numerical subscripts, e.g. E 1, H3, etc., refer to the sequence of adsorption, customarily from the top downward, of respective epiphasic (E) or hypophasic (H) carotenoid fractions from a petroleum ether solution on to a chromatographic powder column. (However, should a fraction or fractions pass through the column as filtrates during the primary separation, these receive the primary numerical designations in order of delivery (Fox, 1962).) Thus E h E 4 would signify the fourth coloured zone down from the top of a column, derived from an original epiphasic mixture, which persisted among the epiphasic components after hydrolytic treatment (h). And E h H 1 would refer to a fraction appearing nearest the top of the column, derived from among those components that altered their behaviour from epiphasic to hypophasic after hydrolysis; H h H 4 would designate a hypophasic component, fourth in series from the top, which remained hypophasic (and neutral) after saponification treatment.

METABOLIC FRACTIONATION, STORAGE AND DISPLAY OF CAROTENOID PIGMENTS

9

RESULTS Tissues

A well-pigmented American flamingo, captured from the wild not long before arriving at the San Diego Zoo from Miami, Florida, and given the astaxanthinsupplemented diet thenceforth until its death some 4 months later, was dissected after post mortem examination and its parts were refrigerated and delivered on ice to the laboratory on the same day, where they were again stored under refrigeration awaiting early analysis. The immature testis, liver, spleen, subdermal fat, mesenterial fat, lung parts, adrenals, parts of muscle and portions of naked tarsal skin were placed, each kind of tissue separately, into acetone in stoppered flasks and stored under refrigeration to permit leaching of the carotoneids with exclusion of phospholipids. Skin of P. tuber

The colourless tarsal scale or cuticle was removed, exposing a thin layer of bright red skin; 1.5 g of this skin readily yielded its rich red pigment to acetone, in which it was stored in the refrigerator. Since the skin-carotenoids proved to be soluble in both petroleum ether and 94-96 per cent methanol, exhibiting no sharp preference for either solvent, no further attempts were made to partition the components at this stage. Instead, the whole of the material was saponified in a 90 per cent ethanolic solution of dilute KOH for 1½ hr in a warm water bath. This operation yielded: (1) a small fraction, persistently epiphasic against 90-95 % methanol (hE), resolvable chromatographicaUy into two sub-fractions (hE 1 and hE,.); (2) a red, interfacial K-salt of astacene (hi); (3) a hypophasic component (hH), larger than the epiphasic fraction, neutral and therefore extractable in petroleum ether from the diluted alkaline liquor. The large hH fraction, when shaken vigorously with aqueous neutral systems, yielded a purplish interfacial aggregation, composed of astacene and derived from the earlier hydrolysis of its K-salt. This difficulty was overcome by increasing the concentration of alkali in the aqueous phase (using NaOH instead of KOH) and adding the resulting interfacial red soap to the main fraction of hi. Both the free astacene and fraction h H were hypophasic in 95 or even 90% methanol, giving red-orange solutions in some contrast to the yellow-orange colour in petroleun ether. The chromatogram of hE afforded a minor yellow filtrate (hE1) and a red zone (hE2), eluted as a travelling yellow-orange band by adding a little methanol to the petroleum ether. A little pinkish material remaining near the top may have represented a trace of astacene, but was too small in quantity for recovery. The absorption curves of hE 1 (maximum at 446 m#, inflexion at ca. 475 mtz) and of hE 2 (single maximum at 463 m/x, with slight shoulder at ca. 475 m#) are shown in Fig. 1. The spectrum of hE 1 recalls somewhat that of a-carotene in hexane, but resembles also that of the cis-isomer of neo-V-dehydrocarotene I (Karmakar &

10

I). L. Fox

Zechmeister, 1955), while the profile of h E 2 appears to bc morc like that of dehydrocarotene II, obtained by treatment of/3-carotene with N-bromsuccinimide. It is by no means inconceivable that dehydrocarotenes might be generated as by-products of dehydrogenase action by the intestinal microflora, shown to be capable of chemically altering various substrates, including astacene. Also, it is not impossible that intestinal or serum enzymes may produce chemical changcs in dietary carotenoids.

01'

........

36

---7.. . . . . .

C ZO

.....

If~

D ~ . . . . . .

.io

0 4

~

-

-

.........

....

(;5

hE2

hEl

mr.

FIG. 1. Persistent epiphasic carotenoid fractions from tarsal skin of P. ruber. Solvent : petroleum ether.

Chromatography of the h H fraction yielded two zones in petroleum ether alone: a broad, deep magenta-coloured band at the top of the column and a narrow red band just beneath it. Development with additional petroleum ether now containing traces of methanol produced three fractions: h i l l , a reddish-pink zone remaining at the top; hH,~, a broader red band beneath this; and h H 3, a narrower, pale orange band which migrated through as a filtrate. Fractions h H 1 and h H z were then removed mechanically and separately from the column; h i l l , not eluted by eithcr methanol or acetone, was finally desorbed with the use of glacial acetic acid. Fraction h H 2, giving a yellow colour in petrolcum ether solution, bleached rapidly upon the addition of glacial acetic acid, retaining so little (yellow) colour as to preclude spectrophotometric analysis. Treatment of h H t with alkaline methanol, then shaking with excess saturated NaCI solution and petroleum ether, yielded some red soap at the interface and a pale yellow overlayer of petroleum ether. The red soap was doubtless residual traces of astacene which had not been recovered in the earlier treatment.

METABOLIC

FRACTIONATION,

STORAGE AND DISPLAY

OF CAROTENOID

PIGMENTS

11

The spectral absorption profiles of hH 1 and hH 8 are shown in Fig. 2. The single absorption maximum of hH8 at 455 mix, with a sloping inflexion between shorter wavelengths (450-430 mix), failed to identify this fraction with any carotenoid known to us. In hH~, the maximum at 470 mix, with a slight shoulder at about 480 mix probably reflected the inclusion of the astacene residue referred to above. An additional unidentified fraction was present, however, as evidenced both by the chemical treatment revealing a neutral xanthophyll and by the asymmetrical shape of the absorption maximum. The small amount of yellow xanthophyll was not further characterized. ,0 . 3 0 4 7__../_ 0 ,

........

,

"'~ A

0"8

0-

,o

....

i

0.6

:

1 - - 0 1 5 - hHI ........... h x 3

I 400

,

i

,

,

I 450

.

.

.

.

1 500

,

rnlJ

FIC. 2. Neutral hypophasic fractions from tarsal skin of P. tuber. Solvent: petroleum ether. From another specimen, recently captured from the wild but dead from shock, a piece of the bright vermilion skin from the tarsus was weighed (120 mg), extracted and processed for the recovery of astacene, which was then measured spectrophotometrically, using the optical density of its measured solution at 475 mix. Using the specific absorption coefficient of 170 (determined in this laboratory with the use of crayfish astacene), the value for the astacene concentration in the skin (as astaxanthin) was close to 13.3 mg/100 g of fresh skin. However, a similar skin sample (258 mg) from another bird suffering a similar death, extracted and measured in an identical manner, gave a higher value, 53.9 mg/ 100 g (in astacene equivalents), for the sum total of carotenoids present, or about fourfold the value for astaxanthin alone. In order to confirm the identity of the acidogenic carotenoid fractions from skin and from feathers with that recovered from the crayfish, the three separate products were subjected to comparative and triple chromatography on MgOcelite columns. The Na-soaps from each of these sources were carefully prepared

12

D.L. Fox

in the usual manner, transferred to petroleum ether as the liberated acid, washed, and aliquot volumes of each adjusted visually to give the same depth of colour, and chromatographed upon the powder columns. Each of the three separately, as well as solutions of all three in equal parts, gave identical chromatograms as follows: 1. From petroleum ether alone, a sharply defined fairly narrow, magentared coloured band remained at the top of the column. 2. After addition of methanol traces to the petroleum ether, the magenta-red colour changed at once to a rose-orange; the band widened and migrated slowly downwards while releasing a very faint yellow-orange fraction which preceded it and shortly vanished. Complete spectral identity was established between astacene recovered from skin and from feathers of the American flamingo. Skin of P. chilensis

The bright, opaque, red skin from the heel or leg-joint of the Chilean flamingo yielded a rich orange extract in ethanol. The petroleum ether solution of the whole pigment proved to be nearly all epiphasic against 95°'o, and completely so against 90°'o methanol; this was in contrast to the hypophasic behaviour of the extract of shank-skin from the American species. Treatment of a portion of the whole extract with warm alcoholic NaOH gave, on subsequent dilution, an interfacial red Na-soap, a minor fraction of persistently epiphasic pigment, and a very small hypophasic component. The free carotenoid acid, liberated from its soap by the addition of a little acetic acid, migrated readily into a fresh layer of petroleum ether. To consider the various operations and fractions in order, attention is drawn first to the chromatographically separated components of the raw, whole extract merely after its transference to petroleum ether. This gave a deep pink-rose zone on the MgO-celite column, very similar to the picture given by crayfish astaxanthin which was chromatographed concurrently for comparison; the adsorbed crayfish pigment, when washed with benzene, however, left the adsorbed zone now a salmon-pink in colour, whilst similar treatment of the flamingo-skin chromatogram converted the conspicuous corresponding pink-rose zone to a kind of mauvepink (El) and released a pale yellow component which passed through the column as a yellow filtrate (E~). The filtrate, transferred to petroleum ether, revealed an absorption curve with a minor inflexion centering at 420 mt~, a maximum at 451 m/z and a sloping shoulder from ca. 455 to 465 m/~ (Fig. 3). This fraction remained epiphasic after treatment with alkali. The residual fraction (El) , after elution and return to petroleum ether, absorbed light along a symmetrical, rounded profile with a single, broad maximum centred at 468 m/z (Fig. 3). When transferred to pyridine and compared with crayfish astaxanthin in the same solvent, it gave an identically appearing curve with centre of the single, rounded maximum at 491 m~ (Fig. 4).

M E T A B O L I C

F R A C T I O N A T I O N ,

0'1,

,

S T O R A G E

•

,

,

A N D

,

i

D I S P L A Y

l

i

,

O F

i

,

C A R O T E N O I D

,

,

P I G M E N T S

,

451

d~5~

i' i

i

i

i

I

i

i

i

i

i

i

J

|

i

i

i

m/a

Fxe. 3. Curves of epiphasic carotenoid fractions from heel-skin of P. chi/.e~/s. Open circles : Ex, red solution in petroleum ether; full circles: E~, yellow solution in petroleum ether. 0'4l

•

,

i

,

,

,

i

i

49O i

i

,

I

I

I

0'l

I.l

FIO. 4. Astaxanthin from shell of crayfish. Panulirus interruptus (upper) and from heel-skin of P. chilensis (lower). Solvent: pyridine.

13

14

D.L. Fox

Fraction E 1 became hypophasic after hydrolysis, hence initially was an ester; its now acidic character was emphasized by its ready formation of a red Na-salt at the petroleum ether-aqueous interface and by the transient pink flush exhibited when the liberated compound, dissolved in petroleum ether, was shaken with water. Its chromatogram was similar to that manifested by crayfish astacene, and a mixed chromatogram of the two carotenoids retained their close similarity, i.e. a deep magenta zone at the top of the column in a petroleum ether system; not easily eluted with benzene, but giving, on the addition thereof, a secondary diffuse reddish zone further down the column. "['he chromatographic matching between the Chilean flamingo skin product and authentic astacene was further confirmed by the close spectral identity of the two. The liberated acid from E 1 showed a closely similar absorption curve in pyridine (maximum at 490 m/x), while its petroleum ether solution absorbed maximally over a rounded profile with centre around 466-470 mp., thus behaving very like astacene. The neutral hypophasic product of hydrolysis (EhH) gave a very irregular absorption curve, probably a composite resultant from the presence of two or more components in too small quantities for successful resolution, but exhibiting a general region of absorption between 445 and 468 m/z. Astaxanthin would appear to be a prominent carotenoid in the leg-skin of both flamingo species examined, but is present in relatively higher concentrations and greater proportions in the Chilean bird, and is, moreover, esterified therein. There was no opportunity to make critical comparisons of the minor fractions of persistently epiphasic components in the two species. It may be said only that, in each instance, unusual spectra characterized such fractions, and that these were reminiscent of certain dehydrocarotenes derived by Karmaker & Zechmeister (1955). Regarding the relative proportions of hypophasic carotenoids in the legskin of the two species, it was readily apparent that the American flamingo yielded greater amounts of the neutral xanthophylls than did the Chilean, that the latter stored far more astaxanthin in the skin, but none as such in the feathers.

Blood Notwithstanding the finding of conspicuous astaxanthin in both skin and feathers, this carotenoid was never encountered in any of the othcr tissues examined, including the blood itself, savc fl~r suspected traces in one instance to be mentioned below. This despite the richness of the blood plasma and of many other tissues in carotenoids, e.g. adrenals, liver and visceral fat. Whole blood. Certain abbreviated observations were possible upon carotenoids extractable from the blood-filled shafts cut away from the barbed portion of pin feathers. Such material, taken from a recently dead American famingo, gave orange-yellow acetone or ethanol extracts and a bright yellow colour to petroleum ether, which yielded its pigment largely to 96% methanol and exhibited no trace of astacene by the usual test. The exploration was repeated on pin feathers drawn

METABOLIC FRACTIONATION~ STORAGE AND DISPLAY OF CAROTENOID PIGMENTS

15

from living birds of each species 4 or 5 hr after the flock bad been fed. The naked, blood-filled shafts were cut with scissors from the barbed portion and were dropped at once into alcoholic KOH. After heating each system to assure completion of hydrolysis, the liquor from each of four separate sets of treated shafts was filtered to clarity, diluted with water (containing dissolved NaC1 to minimize emulsification), and shaken with petroleum ether, which assumed a yellow colour, leaving muffs of pale soaps at the interface. These muffs were of a dirty greenish colour in two samples from American flamingoes and in the single one from a Chilean bird, but in a third sample from an American specimen an orange-coloured interracial muff appeared. It was recovered, driven into petroleum ether by treatment with that solvent containing acetic acid; the solvent was washed to neutrality, evaporated and the residue dissolved in a little pyridine to give a pale red-orange solution. While the pigment was present in too small an amount for spectrophotometry, its characteristic behaviour strongly suggested the presence of astracene, and thus of traces of astaxanthin in the blood of the feather-quill. In a more searching experiment, blood was collected from the wing-veins of four healthy American flamingoes. The striking colour of the plasma was due largely to carotenoids, although a trace of laked haemoglobin was also present; soft absorption shadows appeared at 542 and 578 mg, characteristic of oxyhaemoglobin, while an additional absorption shadow was evident at 490 m/x from the carotenoids. Erythrocytes. The separated red cells were washed free of plasma in 0.85o./o NaCI solution; they were next treated with an excess volume of ethanol, and were finally diluted with water and shaken with a little petroleum ether. To this solvent there was yielded a trace of yellow colour, believed to be due to the presence of minor contaminating clots involving plasma. The erythrocytes themselves appeared to be quite without carotenoids. Plasma. A total volume of 15 ml of plasma plus heparin solution (actually involving but 10 ml of original plasma) was treated with ethanol and with petroleum ether, which gradually extracted a golden yellow carotenoid mixture. Its spectrum in petroleum ether followed a smooth, rather rounded, single maximum at 470 m/~, and indicated an original concentration of about 0.97 mg/100 g of plasma ("astaxanthin equivalents"). A chromatogram of the petroleum ether extract yielded no coloured zones to an upper column of CaCO3, but in the MgO-celite column beneath this it left a pink zone, while a yellow to orange band travelled slowly through to yield a yellow-orange filtrate. This filtrate comprised an epiphasic and a hypophasic fraction, while the remaining sorbate, eluted with methanol in petroleum ether, proved to be all hypophasic. Partition of the original petroleum ether extract yielded a large hypophasic portion, preferentially migrating into 95% methanol and a small epiphasic fraction remaining in petroleum ether both before and after hydrolytic treatment, which yielded no acidic carotenoid.

16

I). L. Fox

Chromatography of the persistent epiphase yielded two fractions: EhE., which migrated slowly through the column as a yellow-orange filtrate, and a pink zone adsorbed to the column but eluted when traces of methanol were added, and passing through as a minor yellow fraction E h E I. This component displayed an absorption maximum at 450 mt~ and an inflexion at 465 m/z, while EhE~ showed a profile not dissimilar, save for a secondary maximum at about 427 m/x. Neither of these epiphasic carotenoids thus matched a currently known compound, although their spectra are suggestive of oxidized derivatives of carotenes. They were too small in relative quantity to affect the profile of the petroleum ether solution of total plasma carotenoids. Like the epiphasic fraction, the hypophasic portion also failed to yield any perceptiblc traces of red Na-soap after hydrolytic trcatment. Blood from the Chilean flamingo yielded but very little carotenoid material, which was preponderantly hypophasic. The spectral absorption of the epiphasic fraction exhibited maxima at 443, 425, 400 and 375 m/x, comprising a completely unfamiliar profile and very likely reflecting the presence of more than one constituent. Like other hypophasic fractions from this species, that from its blood displayed a rather irregular curve, tending toward a rounded maximal region, but exhibiting a "two-spiked" peak at 463 and 470 m/x, probably indicating the presence of two or more components. In view of its paucity, the matcrial was not further investigated by chromatography or hydrolysis. Other tissues

Of the other special organs or tissues tested, e.g. liver, bright-orange adrenals, yellow-orange visceral or subdermal fat, pancreas, spleen, lung, muscle, testis (small, pea-sized, cream-coloured and immature) or ovary (small, pale yellow, immature, with many small follicles), none yielded red carotenoid acid soaps by the standard test ; hence none contained astaxanthin or other acidogenic carotenoids. However, much other carotenoid material was extractable, notably from liver, adrenals, fat and spleen, but far less from muscle, lung, pancreas or immature gonads. I.ike the plasma, the spleen, pancreas, lung and muscle yielded a fair hypophasic fraction. Liver, adrenals, testis and fat contained very little hypophasic but chiefly epiphasic carotenoids, whose partitional behaviour was unaltered by hydrolytic treatment. Because their behaviour appeared to be closely similar on partition and on preliminary chromatography, and since individual quantities were very limited, the persistently epiphasic carotenoid extracts from adrenals, visceral fat, testis, pancreas, lung and muscle were pooled for chromatographic separation. The chromatogram of the above common solution of carotenoids from the several tissues was matched qualitatively by a separate chromatogram of the adrenal epiphasic carotenoids alone. Five coloured bands were exhibited: E E h E 1 a rose-coloured band at the top of the column, followed by N-,EhE 2 (pink), EEhE.~ (pale pink-orange), I~EhE 4 (pale yellow) and Y.EhE 5 (pale orange). Of these five fractions the first, third and fifth were recoverable in sufficient quantities for

METABOLIC FRACTIONATION, STORAGE AND DISPLAY OF CAROTENOID PIGMENTS

17

spectrophotometry, and are charted in Fig. 5. ~ E h E I shows a fairly symmetrical curve with a broad, rounded maximum centred at 455 m/z; Y.EhE 3 has a very minor maximum at 470, a more conspicuous one at 442 and a sharp rise at 435 m/z, suggesting generalized absorption toward the shorter wavelengths, such as characterizes numerous so-called chromolipids; finally Y~EhE5 displays a pair of well marked maxima, at 475 and 445 m/z .respectively, thus being not unlike one of the dehydrocarotene I isomers obtained by Karmakar & Zechmeister (1955), but also reminiscent of a-carotene. lo

1

. . . .

i

1

445..

0.5

•

sO0

[hE (BL,CCO) ~[~h[ $ fTISSUES) 4~0

\ ~0

m,u

FIG. 5. Carotenoid fractions from blood and from internal tissues of P. ruber. Solvent: petroleum ether. The small, pale yellow ovary, some of the orange-coloured subdermal fat, as well as liver tissue from another American flamingo were made available for a careful re-test for the possible presence of astaxanthin. The yellow-orange ovarian extract and the carotenoid-rich extracts from fat and from liver each failed to reveal any traces of astacene after the usual procedure. The tough inner membrane lining the crop was bright yellow in colour, and readily yielded a rich yellow solution in ethanol. However, no carotenoid material whatever was recoverable from the ethanol by diluting, acidifying and shaking with petroleum ether. The yellow pigment, remaining in the aqueous phase, faded while standing so was not identified. Egg

A fresh egg was made available since the parents had abandoned it 3 days after a rain. It had a matt-white, chalky appearance, measured 90.5 mm (major axis) by 54-0 mm (minor axis), weighed 146 g, and revealed a faintly blue-grey shell beneath the chalky exterior when some of this was scraped away. The interior

18

D.L. Fox

walls of the shell exhibited a very pale greenish cast. The colourless albumen and bright orange-apricot-coloured yolk weighed 128 g in the aggregate, the separated yolk 34 g (cf. Allen, 1956). An acetone extract of the yolk was of a rich orange colour, and, when filtered through celite, left white, coagulated protein and presumably phospholipid materials. The carotenoids were transferred to 275 ml of petroleum ether. The pigments did not separate between petroleum ether and 90% methanol; rather, the pigments were ambiphasic in such treatment, but with preferential hypophasic behaviour. However, the portion which ultimately remained almost entirely epiphasic against 85% methanol was separately treated and referred to as the epiphasic fraction. Preliminary spectrophotometric examination of (1) the whole yolk extract, (2) the epiphasic and (3) the hypophasic fractions, each in petroleum ether, yielded the following results. The combined pigments, showing a rounded maximum with centre at 465 m/z, revealed an approximate concentration of 2.24 mg (in astaxanthin units) per 100 g of yolk material. The epiphasic portion gave a maximum at 452 m/z and an inflexion at about 485 m/z. The whole hypophase showed a single peak at 455 m/z. Chromatography of the epiphasic pigments resolved two fractions: E 1, a diffuse, rose-coloured zone extending downward from the top, and E2, a narrower pink to red band which travelled slowly through the column as a filtrate. The residual zone E 1 was eluted by methanolic petroleum ether, travelling through the column as a narrow orange band. The absorption spectra of these two fractions are plotted in Fig. 6, E 1 showing a rounded contour with centre at 460-462 m/z, while E 2 has a sharp peak at 450 m/z, an inflexion close to 425 m/z, and a singular, sharply defined shoulder from 465 to 471 m/z. Subsequent hydrolysis of each fraction revealed pale soaps at the interface, but no astacene. A faintly coloured product from the soap E~I in the hydrolysate of E 2 proved to be a fatty acid, and exhibited an absorption curve without maxima but rising toward the region of shorter wavelengths, thus not characteristic of carotenoids. Following the chromatographic separation and before subsequent hydrolytic treatment, each of the fractions in a small volume of petroleum ether was chilled overnight to about -- 2WC, whereupon snowy white flakes of lipid materials were deposited and recovered. The collective lipids from E 1 gave a positive test fi)r unsaturated sterols (Liebermann-Burchard reaction), displaying a sequence of pink, violet and deep blue colours. The material from Ee gave no response to the same test, so contained none of the unsaturated sterols. After hydrolysis of E2, and befi)re spectroscopy of the resulting persistently epiphasic material (E,,hE), the latter was likewise chilled (to about --25C), and yielded organic acids free from unsaturated sterols. These lipid fractions have been saved, but have received no further study. Fraction E l showed unchanged phasic behaviour after hydrolysis, while E.~ so treated then yielded a little more hypophasic material than before, including the coloured soaps of organic acids. Its absorption spectrum in carbon disulphide

M E T A B O L I C F R A C T I O N A T I O N , STORAGE AND D I S P L A Y OF CAROTENOID P I G M E N T S

19

showed a single peak at 482 m#, inflexions at 500 and at 520 m/~, failing to match any carotenoid known to us. Echinenone (~= myxoxanthin) has its maximum at 488 m# in carbon disulphide, and its etherel.solution develops a greenish-blue colour when shaken with concentrated HCh The fraction E~hE failed to resemble echinenone either spectrally or in its lability toward strong HCI. 0%

a

01

ol

F~c. 6. Absorption.spectra of epiphasic fractions Et and Ez from P. ruber egg-yolk. Solvent: petroleum ether. The hypophasic portion yielded but one carotenoid on chromatography. A feeble and diffuse yellow non-carotenoid component (Hx) adsorbed in the lower third of the column gradually filtered through on the addition of more solvent; this material absorbed light gradually and increasingly toward the violet end of the spectrum in the same fashion as did fraction E~hl. A second filtrate, eluted by methanolic petroleum ether, was colourless and yielded, upon evaporation of the solvent, a white deposit of fatty material with an odour very like that of turkeyfat. The final fraction/-/3, a diffuse pink zone, migrating down the column as a narrow pink-orange band on elution with methanolic petroleum ether, presented a symmetrically rounded peak at 458 m/~, in petroleum ether, reminiscent of canthaxanthin (Fig. 7). Following hydrolytic treatment, this fraction, now H~hH, light yellow in petroleum ether solution, exhibited an absorption curve of the same shape as before (maximum 460 mtz) and, at the same concentration, gave a salmon-pink colour to pyridine, wherein its maximal absorption was at 488 m/z (cf. Manunta, 1939) and at an intensity (height) of about 23 per cent beneath that at the 460 m/x peak in petroleum ether. The pyridine solution exhibited a second and very high

20

I). L. Fox

peak at 302.5 m/z (Fig. 8). No acidogenic carotenoid, e.g. astacene, was detected in the hydrolysate. Despite the similar shapes and maxima of H a (458 mtL), H:~hH (460 mt~) and E 1 (462 mt~), the hypophasic materials migrated out of the petroleum ether and into 90% methanol (cf. canthaxanthin), while the epiphasic remained ~ilmost entirely in the petroleum ether layer, even after hydrolytic treatment. 0.8[

p

I

I

I

I

I

458

0.6

0'4

0'2

I

300

,

I

350

,

1

I

400

450

,

i

1

I

500

550

600

rnju FIe,. 7. Absorption curve for fraction//3 from P. ruber egg-yolk. Solvent: petroleum ether. Like most tissues, the egg-yolk revealed no accumulation of astaxanthin, but yielded mainly atypical, usually single-peaked carotenoid fractions suggestive of oxidative (dehydrogenated) products. Canthaxanthin was suspected as well. Faeces

of P.

ruber and

of P.

chilensis

The examination of intestinal contents is considered next because of the manifest destruction of dietary carotenoids in the gut. It had been observed repeatedly that the fluid faeces voided by either of the captive species never appeared red or yellow, but white or very pale in colour. In view of the distinct pink and orange

METABOLIC FRACTIONATION,

STORAGR AND DISPLAY OF CAROTENOID

PIGMENTS

21

colours of the raw, mixed food (due largely to the presence of astaxanthin and other carotenoids), the colourlessness of the flamingoes' faeces suggested an efficient agency for chemical destruction of carotenoids within the gut. A few chemical and microbiological experiments emphasized this hypothesis. 0'3,

302.5,

I

I

I

i

I

I 500

t 5~

6~

/-,, 0.25~ I I I I 1 300 305

480 0.2

0.1

0-05

I ~0

I 3~

I 4O0

! 4~ m,O

Fro. 8. Absorption profiles of fraction H3hH from egg-yolk. Closed circles: light yellow solution in petroleum ether. Open circles: pale salmon-pink solution in pyridine. Same concentration in each solvent. From the gut of an American flamingo which expired from shock following pinioning operations, we recovered copious masses of white, limpid, and light brown material. The ethanolic extract of the portion from the small intestine was bright yellow, while that from the material taken from the large intestine was nearly

22

D.L. Fox

colourless, and its filtrate, after dilution and extraction with petroleum ether, yielded only feeble traces of yellow colour. The ethanolic system containing the material from the small intestine was hydrolysed with NaOH, then diluted with concentrated NaC1 solution, and shaken with. petroleum ether, but yielded only barely perceptible traces of yellow pigment to this solvent, while the interface exhibited only a dirty yellow muff. The yellow colour of the original ethanolic extract therefore could hardly have been due to carotenoids. The practically colourless extract of the material recovered from the large intestine was not further processed. The body of a Chilean flamingo, refrigerated and examined on the day of its death, yielded green, brown and pinkish material from its intestines. Treatment of the whole material with ethanol resulted in a pale yellowish-green solution. Again, dilution of the filtrate, followed by extraction with petroleum ether, yielded only faint traces of vellow pigment, which proved to be hypophasic. In both species of flamingo examined, nearly all of the copious dietary carotenoids remaining in the gut had been chemically altered. Hence carotenoids reaching skin, feather follicles and other tissues via the blood must represent the cumulative stores of minute assimilated quantities. Biochemical activities in the gut of P. tuber

From the above observations it was suspected that active dehydrogenaseproducing bacteria might be encountered in the flamingo's gut. An opportunity to explore for these was afforded by the death of an American flamingo following the accidental fracture of a wing. The entire gut, from oesophagus to cloaca, was removed, refrigerated, and, on the day of the bird's death, delivered in an icepack to the laboratory where it was again stored in a refrigerator overnight. Aerobic and anaerobic cultures of micro-organisms from both small and large intestine were prepared separately by Dr. Richard Y. Morita, who tested them for dehydrogenase activity using Thunberg tubes and methylene blue as a hydrogen acceptor. Dehydrogenase activity was demonstrated in both aerobically and anaerobically cultured fi)rms, each from both small and large intestine, in the presence of M/50 concentrations of the following substrates: acetate, malate, glucose, c,-glycerophosphate and fi)rmate. Succinate as a substrate gave feeble or questionable results in all four instances, while lactate showed a positive response for dehydrogenase action in all save the anaerobic culture from the small intestine. E. coli was found in the small intestine, but no sulphate reducers were encountered in an 3, part of the gut. In an additional experiment, astacene, isolated from feathers, was exposed to anaerobic micro-organisms from the flamingo gut, under nitrogen gas at 30°C in a dark cabinet. Aliquots taken after a few days showed the now orange (not red) pigment to be largely adsorbed to solid materials in the culture, while the pigment in the sterile control remained a red-orange colloid. After a week, both the cultured material and the control yielded only acidic fractions, as originally. However, while the control still yielded its carotenoid to petroleum ether, showing therein

METABOLIC FRACTIONATION, STORAGE AND DISPLAY OF CAROTENOID PIGMENTS

23

the familiar single rounded maximum close to 472 m/~ (astacene), the culture failed to yield any pigment to petroleum ether but conferred upon pyridine a peachcoloured solution with a very broad and irregular absorption curve showing a centre of absorption at about 480 m# (not 490 m# as by astacene in pyridine). Clearly there are, at least in captive flamingoes, dehydrogenases in the gut which may contribute to oxidative operations, probably including chemical changes in caroter~oids. Although the carotenoid-free condition of the material within the alimentary tract would suggest the primary role of micro-organisms in the destruction of the pigments, it is possible that the serum or the intestinal mucosa may contain enzymes for the alteration of certain carotenoids (cf. carotene conversion to vitamin A in mammals and the mucosal barrier against xanthophylls in the horse, cf. Zechmeister et al. (Fox, 1953) and apparently against all save fl-carotene in the marine polychaete Thoracophelia mucronata (Fox, Crane & McConnaughey, 1948)). Chick of P. ruber Healthy chicks, hatched in captivity, exhibited the whitish or very pale grey, downy plumage, black eyes, straight red bill and swollen, bright red legs and feet, all of which features characterize the young at this early stage (Gallet, 1950). One chick's weight 48 hr after hatching was 149 g, according to K. C. Lint. The podgy little legs and feet, barely able to support the weight of the body, exhibited fat creases, dimples and a soft flesh-pink colour. The young bird's legs and feet, growing thinner with contraction of the skin, remained bright red in colour for at least its first week of life, during which it remained upon the nest, was fed by each parent, and showed rapid growth. On about the eighth day it left the nest, by which time the legs had blackened (K. C. Lint, personal communication). Certainly the legs and feet were still bright red at the age of 1 week, but a week later only the distal portion of the bill and the soles of the feet remained pink; the exposed skin of legs and feet appeared sooty black. At the age of 16 days the chick was killed by a single sharp blow on the head, report.edly by a horned screamer. This, however, made it possible to examine carotenoids from certain internal organs and tissues and a small piece of skin excised from the tarsal joint by the museum preparator before he proceeded with the mounting. No recognizable traces of carotenoid were found. The downy plumage was grey and white, but nearly black at the base; the short bill, pale in colour, with pinkish and dark greyish areas, had become slightly curved downward, ending in a sharp tip on the upper mandible. The eyes were jet black instead of being pale yellow as in the adult bird. The body-skin beneath the feathers appeared fleshy pink, as in common fowls. The integument covering the tibiotarsal, tarsal joint, shank and dorsal surface of the toes was a shiny black or very deep grey, showing in some parts very thin, transparent sloughing fragments of epidermal origin.

24

D.L. Fox

The heel-skin, normally bright red in adults, showed in the instance of the chick, even on microscopic inspection, no traces of red or yellow pigment, but only an epidermal layer of dark grey colour and an underlying dermal stratum of matt-white appearance. Moreover, the tissue yielded no traces of carotenoid pigment after standing in darkness for several days, immersed in a small volume of methanol at refrigeration temperatures. The subdermal fat was very pale. The heart and contained blood, all finely comminuted under ethanol in a blender and subsequently stored under refrigeration, yielded none but traces of pigment. Similarly, the visceral organs yielded only traces of yellowish-coloured material. The gut itself was separated, emptied of its food contents (which yielded a little pigment, as expected) and, extracted separately, gave a small quantity of pale yellow material. In view of the great paucity of coloured material recoverable from the various sources, the alcoholic extracts of all the internal organs were pooled, transferred to petroleum ether and chilled to ca. - 18~C overnight in order to precipitate solidifiable lipids. After filtration of the resulting cloudy precipitate, the pigment solution, given the partition test with 9()c!,, methanol, was found to involve nearly completely epiphasic pigments. Hydrolysis in ethanolic NaOH produced a deepening of the colour from pale yellow to yellow-brown, and rendered the coioured material hypophasic. Addition of concentrated NaCI solution and a little petroleum ether produced a faintly murky yellow interfacial muff of soaps, but gave no coloured component to the petroleum ether. Recovery of the liberated acidic component in petroleum ether and spectrophotometric examination of this revealed no maxima, but only a steadily rising absorption curve, characteristic of chromolipoids or their organic acids. No carotenoids were detected in the melanized skin of this chick, nor in a comparable sample from the pink, non-melanized leg-skin of a second chick, which had been accidentally trodden to death at the age of 4 days. Finally, an unstained piece of the heel-skin was fixed in paraffin, sectioned and mounted fi)r high-power phase microscopy. No pigment save dark melanin could be detected. Further sections were cut, mounted and photographed, clearly demonstrating the disposition of dark aggregates and spheroids of melanin, from deep portions out into the exposed later of epidermis (Fig. 9). Feathers

There are wide differences in ratio of white to pink vermilion areas of plumage and in the depth of pigmentation, not only between individual birds but among the plumes and even in individual plumes of single birds. However, there was no doubt as to the general striking pink appearance of the San l)iego flock's new feathers after several months' administration of the astaxanthin-rich diet. The contrast with the birds' earlier paleness was further apparent in coloured photographs taken of the same flock before initiation of, and after a year on, the experimental diet. First-hand views of P. ruber in flocks at the Copenhagen Zoo and at the New York Zoo, supplemented by gifts of flight feathers from both sources, have

FIG. . Microscopic section of black or deep grey skin from tarsometatarsal joint Ieel) of 16-day-old American flamingo chick ( x 840). Note heavy concentratic )n of large melanin aggregates in dermis and small, rounded melanin granules in eplid.ermis. (Paraffin mount by Miss Thea Schultze; section and photograph by Dr. Howard Bern.)

METABOLIC

FRACTIONATION,

STORAGE

AND

DISPLAY

OF CAROTENOID

PIGMENTS

25

afforded an appreciation of the close similarity in feather pigmentation between those birds and the same species kept in the San Diego Zoo. This notwithstanding the very different kinds of carotenoids fed at the three respective, widely separated sites, i.e. (1) capsicum (paprika), (2) carrot oil in alfalfa meal, sold commercially as "Caradee", and (3) ground crayfish shells plus red salmon flesh. Moreover, chemical analyses have revealed close qualitative similarities between the set of carotenoids stored in the feathers of all three colonies, but with minor differences in ratio between the principal component canthaxathin, astaxanthin and lesser fractions. O0VerN I cartl~ t t ~t e ~

~oumt

--~sel

kx~me

c ~ . ~ berc~ ~er rod or m~ cg,~md ~ t

~t

FIc. 10. Diagrammatic sketch of the cross-section of a feather barb from the American flamingo, Phoe'nieo'pterus tuber.

The enclosure of the carotenoid material entirely within the barbs and central rachis of the feather explains to some extent the failure of the common fat-solvents to remove the pigment. Finely cut, thin, unstained microscopic cross-sections of barbs show the pigment's encasement within the keratinaceous walls (Fig. 10). Moreover, very little lipid material seems to accompany the carotenoid deposited there. Well-pigmented flight feathers of combined weight 3.9 g were divested of the naked, hollow, white portions of their quills and were then extracted of any adhering preening oils or other adventitious fat-soluble detritus by treatment in a Soxhlet apparatus with refluxing diethyl ether and ethanol (2:1; v/v) for 7 hr without the leaching of any carotenoids. Subsequently, non-saponifiable lipid materials, extracted with petroleum ether from an alkaline hydrolysate of the feather material, amounted to but 0.0370 g, while the combined organic acid fraction, similarly extracted from the now mildly acidified system, aggregated a similar value, 0.0323 g. Neither extract yielded the coloured sterol reaction in response to the Liebermann-Burchard test. The red colour of the feather carotenoids in situ, the not surprising low lipid content of the feather material, and the failure of cold or warm acetone, ethanol or diethyl ether to dislodge the coloured molecules even from cut feathers, all suggest that the carotenoids are in some fashion bound to the keratin to give a solid chromoprotein. The keratin's immunity to customary coagulating or denaturing reagents such as alcohol or acetone is in keeping with its general insolubility, while alkalis readily digest the keratin, releasing the heretofore bound carotenoids; and pyridine, an active penetrant of solid proteins and a successful

26

D.L. Fox

solvent for many others, dissolves away the "conjugated" carotenoid material, which thereupon changes from red to orange in colour. Allen's report (1956) that "a very red flamingo f e a t h e r . . , placed in a jar containing preserving alcohol turns completely white within a few minutes" remained unverified in these studies. Glacial acetic acid extracted only a part of the pigment during an overnight interval and the boiling reagent caused bleaching. Pure 2-mercapto-ethanol, H S - - C H 2 - - C H 2 - - O H , caused the feathers to bleach completely during an overnight interval at room temperatures. Aqueous solutions of N%S also destroyed the feather carotenoids, which faded in a day or two at room temperatures from pink, through orange, to dirty brown-yellow. Still another sulphur-containing compound, the amino acid cystine (--S -CH,,-CH(NH~)COOH)2, was destructive to astacene (from crayfish) in the presence of dilute alkali ill ethanol when heated to 58-60'C for 3 hr. Concentrated aqueous LiC1, reportedly used successfidly on carotenoidcontaining feathers of some species, seemed to be without effect upon the subsequent extractability of the flamingo plume pigments by organic fat-solvents. V61ker's method (1954b), involving exposure of the feathers for a week to concentrated aqueous KCNS solutions, gave unsatisfactory results, since subsequent treatment with methanol, while removing considerable yellow material, yielded no astaxanthin, known to have been present originally, and left unextracted pigment within the feathers. Aqueous alcoholic NaOH (ca. 2_~o,/o, w/v, in 85-90 o~ ethanol) digested the feather-keratin, releasing the carotenoids to give orange-yellow solutions. This approach was used in the beginning of these studies, allowing the closed systems to stand overnight at room temperatures, or more commonly gently stoppered to allow the alcohol vapour and much air to escape slowly during warming over a water-bath at ca. 60cC for ~-1-~ hr. After digestion of the keratin, the liquor was filtered through celite to remove colourless insoluble residues, was then diluted with four or more volumes of water, or with concentrated NaCI solution to preclude emulsification, and shaken with a small volume of petroleum ether. Carotenes and neutral xanthophylls were thus driven into the epiphasic petroleum ether layer while the Na salt of astacene, generated by the alkalicatalysed atmospheric oxidation of astaxanthin, appeared as a red layer at the liquid junction. (N.B. Any astacene salt, hydrolysed at the interface despite the presence of the dilute alkali, thus permitting the liberated acid's migration into the petroleum ether phase, was readily recoverable therefrom by increasing the concentration of alkali in the aqueous phase and then re-shaking.) The alkali treatment of feathers proved to be an effective method for detecting and recovering the astacene derivative, but was suspected of altering slightly some of the other carotenoid components, as observed also in studies of the feathers of the scarlet ibis (Fox, 1962). This contingency was emphasized by later comparison with pyridine-extracted feather-xanthophylls; by the observation that alcoholic alkali applied to relatively pure astaxanthin for the preparation of astacene sometimes caused bleaching (although it seemed to remain unchanged if treated in the

MIg'rABOLIC FRACTIONATION~ STORAGR AND DISPLAY OF CAROTENOID PIGMENTS

27

presence of feather keratins) and by recognition of the fact that feather-keratins involve combined, reduced sulphur, thus contributing sulphide ions to an alkaline digest, and introducing a factor potentially harmful to liberated neutral carotenoids. Also, while an experimental extraction procedure involving added Pb(OH)2 as a precipitant of the liberated sulphide compared favourably with the alkaline treatment alone, i.e. omitting the lead, this gave no assurance that some of the carotenoid fractions might not have been altered by the alkali itself, as is the algal xanthophyll fucoxanthin (Strain, 1942). More reliability was placed on the use of freshly distilled pyridine kept in dark bottles. It was found in these investigations that this solvent, warmed on an 80°C steam bath, extracted the carotenoids in about 2 hr, leaving the immersed feathers white or nearly so (Fox, 1962). Mild aqueous ethanolic NaOH could be used for the recovery of astacene and canthaxanthin, and was so applied as an alternative method for those fractions, but pyridine extraction was preferentially applied for recovery of astaxanthin and the other neutral carotenoids, including canthaxanthin. Separate pyridine extracts of feathers from P. tuber at the San Diego, New York and Copenhagen Zoos were transferred by dilution to petroleum ether, therein rinsed free of pyridine, and each batch was chromatographed from the solvent upon MgO + celite. Each system presented at the outset a single thick, bright red zone at the very top of the column. But upon development of the chromatogram by adding traces of methanol to the petroleum ether, each system was resolved into a small series of coloured zones, i.e. four each for the San Diego and New York feathers, and three for the Copenhagen ones. The respective sequence, colour and single absorption maxima in petroleum ether were closely similar, as follows: Fraction number (from top)

San Diego

1

Pale pink: 466 mk~

2 3 4

New York

Very pale pink : 466-467 mp Pale pink: 464 m~ Very pale pink : 464 m/z Deep pink : 462 : m/~ Rich pink : 460 m# Very pale yellow : Pale yellow : 448 m~ 446 m/~ ( ~ 466)

Copenhagen

Pink-orange: 465-466 m/~ Pink-orange : 465 m~ Orange-red: 462 m/z

The relative preponderance of these fractions, inter se, in the different sets of feathers, was as follows: S.D.: 3 > 2 > 1 > 4 ; N.Y.: 3 > 2 > 4 > 1 ; C.: 3 > 2 > 1 . Chromatographic fraction 1 was astaxanthin, as established by its spectral absorption maximum, its convertibility into astacene, and the spectral and chromatographic identity between the latter (from S.D. feathers) and astacene isolated from the marine crayfish Panulirus interruptus, upon the ground shells of which the birds in this flock were fed with their regular food. It should be re-emphasized that alkaline treatment of chromatographically isolated astaxanthin sometimes

28

D . L . Fox

catalysed the oxidation to complete bleaching (e.g. as with small astaxanthin quantities recoverable from the Copenhagen feathers), but that hydrolysis of the whole feathers always yielded astacene. Fraction 2, an ambiphasic xanthophyll remaining neutral after alcoholic alkaline treatment, is believed to have been an isomer of canthaxanthin (fraction 3), with which it is closely similar in spectrum, showing a maximum of from 2 to 4 mix longer wavelength (Zechmeister, 1960). T h e predominant component, fraction 3, an amphibasic neutral xanthophyll, was chromatographically and spectroscopically identified with an authentic preparation of canthaxanthin provided by Professor Zechmeister. T h e canthaxanthin fraction from the San Diego feathers was similarly identified with the corresponding fraction from the New York feathers, and with a similar fraction from the plumes of the scarlet ibis (Fox, 1962). T h e close chemical and spectral similarities between the canthaxanthin fractions from these birds and the corresponding pigment recovered from the Copenhagen feathers left no doubt as to the latter's identity with the same compound. T h e fourth and final fraction, not evident in extracts of the Copenhagen feathers, recoverable in small traces from the San Diego material, but found as the third most prominent pigment from plumes of the New York flock (i.c. in lesser quantities than canthaxanthin, but exceeding astaxanthin), was an unidentified epiphasic compound, showing absorption in petroleum ether with a maximum at 448 mix and inflexions at 420 and 467 mix (Fig. 14). As might have been expected, greater proportions of astaxanthin were evident in the feathers of the San Diego flamingoes, which received substantial astaxanthin-rich supplements in their diet. It was of particular interest to encounter some astaxanthin also in the plumes of the birds receiving diets containing none of this compound, according to the respective curators' lists of fi)od constituents, reported as follows. DIETS OF ('APTIVEI:L.AMINGOF~q A t Copenhagen Zoo (Dr. H. Poulsen)

per cent Herring meal* 6 Skimmed milk powder 8 Pig fat 2 Soya-bean meal 29 Sun-flowermeal 3 Yellow corn meal 5 (;round sorghum 14 Barley meal 10 Wheat bran 8 Wheat germ ( + 2 per cent yeast) 5 Malt 2 Minerals 4 Vitamin sources 2

At New York Zoo (Mr. W. (;. Conway)

Super l.aying Mash Raw ground carrots Boiled ground horse meat 180 D Super Caradee*( = alfalfa leaf meal containing 10 per cent by weight of concentrated carrot oil) Table salt Bone meal

per cent 55 18 14

10 2 1

* The herring meal is reportedly made of whole, dried herring minus its oil. It failed to yield astacene, or indeed any detectable carotenoids. The carotene-rich Caradee likewise yielded no astaxanthin (see text).

METABOLIC FRACTIONATION, STORAGE AND DISPLAY OF CAROTENOID PIGMENTS

29

Dr. Poulsen reports that, on the above diet alone, both the flamingoes and the scarlet ibises fade in colour, hut that this is restored when copious paprika powder is introduced into the food. Mr. Conway achieved the same restoration of feather-pigment in flamingoes in the New York Zoo after supplementing the diet with the "Caradee". (The latter product has, since the completion of the research in the San Diego Zoo, been added to the dietary of the San Diego flock, with resulting increases in feather pigmentation.) Diagnostic tests were conducted for the possible presence of astaxanthin in the carotenoid supplements used in flamingo food at the New York and Copenhagen Zoos. To explore for any traces of astaxanthin in "Caradee" fed to the New York flock, a sample of this carotene-rich food was extracted with absolute ethanol, to which it imparted a deep red-brown colour. This extract was hydrolysed, diluted and extracted quantitatively with petroleum ether for carotenoids. No trace of astacene appeared at the interface of the petroleum ether and the alkaline, aqueous alcohol. Likewise, the paprika powder, constitutuing the carotenoid additive to the diet of the Copenhagen flock, was similarly tested. Its hydrolysate yielded a little orange-red material at the interface on shaking the diluted system with petroleum ether. This acidic fraction, recovered by adding a trace of glacial acetic acid to the petroleum ether, exhibited in the latter solvent, rinsed free of the acetic acid, a maximum at 465 m/~ but, unlike the smooth contour of astacene, definite inflexions ar 447 m/, and 4-93 m/~. Typical spectral profiles obtained with the Carey instrument for fractions from the feathers of the three different flocks of birds are shown in Figs. 11, 12, 13 and 14. The close similarities between determinations made with the Beckman and Carey instruments afford confidence in the earlier work on skin, internal organs and eggyolk, when only the Beckman equipment had been available. Absorption spectra of pure canthaxanthin (from Professor Zechmeister) are shown for comparison in petroleum ether as well as in pyridine in Fig. 15. The petroleum ether system exhibits a profile very like that which characterizes the canthaxanthin fractions recovered from feathers, although its chief maximum at 463.5 m~, its lack of a definite cis-peak at around 360 m/~, and the appearance of a minor peak at 294 m~ suggested some recessiveness in the presence of cis-isomers (Haxo, 1950; Zechmeister, 1960). In view of these findings, and since, according to the respective curators, no crustacean or other known source of astaxanthin-containing food was administered to either the New York or Copenhagen flamingoes, it would appear that the American species possesses in its system the ability to convert other carotenoids partly into this diketo-dihydroxy compound. However, in view of the presence of major quantities of the simpler but even more rare diketo carotenoid, canthaxanthin, in the feathers of this flamingo species and in those of the scarlet ibis, the presence of astaxanthin as well among the end-products is perhaps less remarkable. In view of the flamingo's storage of canthaxanthin, notably in the feathers, it might well be expected that a supplementation of the diet by a rich source of this

30

D . L . Fox 0"4 . . . .

. .......

• ........

d 0.~

FIG. | 1. Astaxanthin from P. ruber feathers, in petroleum ether. (San Diego flock.)

~

O7

C-5

~l

/I

\ \\,,

o~

300

~10

400

4~

ga~

!~0

~

~0

M0

Fie,. 12. Astacene from

P. ruber feathers, in petroleum ether (solid line) and in pyridine (dashed line). (San Diego flock.)

08

07

O~

O~

d

C'~

03

07

DI

0~'

3m

330

~o

l:l(;. 13. Canthaxanthin from

~

~o

3~0

i

~o

P. ruber feathers, in petroleum ether. (San Diego flock.)

METABOLIC

FRACTIONATION,

STORAGE

AND

DISPLAY

OF CAROTENOID

PIGMENTS

31

carotenoid, e.g. the edible pink mushroom Cantharellus dnnabarinus (Haxo, 1950), should emphasize the bird's natural pigmentation. Analyses of flight feathers from the Chilean species, P. du'/ens/s, from both the San Diego and New York Zoos, gave comparable results inter se, and similar in some important respects to the findings on P. ruber feathers, in that both sources yielded acidogenic carotenoid material, accompanied by major amounts of ambiphasic, neutral xanthophylls corresponding spectrally to canthaxanthin, with lesser quantities of an epiphasic component of unique absorption profile. O6

0"$

O~

d 0"3

&2

&l

DO

FIG. 14. Unidentified epiphasic carotenoid from P. tuber feathers, in petroleum ether.. ("Caradee" fed New York birds.) •o

~o

~

~

ot--

o8--

d

o2 s~o

mo

Mu

Fro.

15. Pure canthaxanthin (ex Cantharell~ by L. Zechmeister), in petroleum ether (max. 463"5 my) and in pyridine (max. 488"5 and 303 my).

The P. chile~is feathers, upon direct hydrolysis, yielded a red sodium soap which, on release by acidification, exhibited in petroleum ether an absorption maximum at 454 my and a minor peak at 373 my; in pyridine the material showed

32

D. 1,. Fox

maximal absorption at 474 mtL, with nfinor peaks at 386, 336 and 305 m/~, thus differing from astacene, derived from astaxanthin in P. ruber feathers. The red soap was accompanied by lesser amounts of another residue, transferable only with difficulty to petroleum ether, which was lost. From the petroleum ether solution of neutral carotennids recovered from the feather hydrolysate, an epiphasic carotenoid was obtained whose spectrum matched closely that of a similar fraction encountered in an unhydrolysed extract leached from the feathers with pyridine. Several bright vermilion feathers were soaked overnight in pure pyridine, under nitrogen gas, in a glass-stoppered flask set on top of a water bath warmed to 90'"C. The resulting orange-coloured pyridine solution, diluted and shaken with washes of petroleum ether, yielded all of the pigment thereto. This solution, freed of pyridine, yielded most of its pigment to 95"~, methanol, but left a yellow epiphasic fraction. The epiphase, washed free of methanol and chromatographed upon MgO + celite (1:1), exhibited, on development with further petroleum ether, now containing traces of methanol, a single thin orange band of pigment which, when eluted by addition of more methanol, gave a yellow solution in the washed petroleum ether, and presented therein an absorption maximum at 448-449 m~, with shoulders at 470, 430 and 400, and a plateau from 370 to 360 m/~. This fraction was thus reminiscent of fraction 4, an epiphasic one, from feathers of the New York flock of P. ruber. It spectrum was not recognized as belonging to any known carotenoid. The hypophase of the original pyridine extract yielded four fractions as follows : 1. A rose-red band; broad maximum at 465; inflexion at 360--380 mt~. 2. A rose-red band; broad maximum at 463; inflexion at 360-380 intL. 3. A deeper pink band; broad maximum at 460-461; inflexion at 360-375; minimum at 327 mtL. 4. A narrow orange band; maxima at 447-448 and 370; inflexion at 472 m~. This resembled, in its epiphasic behaviour and spectrum, the epiphase described above; its recovery in the hypophase resulted from its partial yielding to repeated extractions with the 95'~, methanol due to a partitional distribution not confined completely to the petroleum ether phase. Fraction 1 of the above series, given hydrolytic treatment, yielded a small amount of interfacial soap in insufficient quantity for spectroscopic analysis, but was in all probability the same material as was recoverable by direct hydrolysis of the feathers. The chief component of this fraction, and of fractions 2 and 3, was undoubtedly isomerized canthaxanthin. Indeed the third fraction's spectrum displayed an absorption profile identical with the commonest profile of canthaxanthin. An opportunity to examine the plumage of another flamingo, Phoenicoparrus jamesi, revealed similarities between this material and the feathers of the other two species. A specimen of this rare little three-toed South American flamingo, collected in Bolivia by Mr. Roger Peterson, was loaned to Mr. William Conway of

M E T A B O L I C F R A C T I O N A T I O N , STORAGE A N D D I S P L A Y O F C A R O T E N O I D P I G M E N T S

33

the New York Zoo, who has since collected living specimens from high mountain lakes of Bolivia and who kindly sent a few of the vermilion-coloured wing coverts and some long, deeper red plumes, taken from the wild birds, to this laboratory. Unlike the feathers from the American flamingo, Phoenicopterus tuber, or from the Chilean species, P. chilen.tis, this material failed to yield its pigment so readily to pyridine, e.g. when warmed therein at 62°C for 5 hr and at 85°C for an additional hour or more. Exposure to the solvent at 70°C overnight, however, effected the extraction of nearly all of the carotenoids. Diluted and transferred to petroleum ether, all save possible traces of the pigment were extractable therefrom with 95% methanol. The petroleum ether solution of the hypophase from the original pyriciine extract, chromatographed upon MgO-celite (1:1), presented a rose to brick-red zone at the very top of the column. Addition of methanol traces to the system altered the chromatogram to give a descending pink-orange zone, followed by a separate pale pink band. Each of these zones, separated mechanically, eluted with petroleum ether containing a little methanol, filtered, rinsed free of the methanol and examined spectroscopically, exhibited absorption profiles typical of canthaxanthin, i.e. (1) maxima at 461 m/~ and 360 m/~; minirhum at 328 m/~; (2) maxima at 460-461 m/~ and 362 m/~g minimum at 328 m/~. Each fraction was subjected to alcoholic alkaline treatment. The first one retained a neutral component, but yielded also a trace of red interfacial soap, while the second zone yielded no acidogenic material, and exhibited the same absorption as before. Some of the feathers were cut up and treated with mildly aqueous ethanolic NaOH in a warm-water bath. Ultimate filtration of the liquor through celite yielded a red-orange solution which, upon dilution and shaking with petroleum ether, yielded a red soap. This, on mild acidification and transferral to petroleum ether, presented in the washed solvent an absorption typical of astacene: a rounded, symmetrical maximum at 469-470 m/~ and a very minor one at 294 m/~. In pyridine the curve was likewise completely typical: a similar rounded maximum at 490 m/~ and a very minor rise at 304 m/~. Similarities between the carotenoid pigmentation of Phoenic@arrus jamesi plumage and that of the two Phoenic@terus species therefore involve the presence of major amounts of canthaxanthin and minor quantities of aslaxanthin or closely similar acidogenic compounds. These comparisons apply also to the feathers of Guara rubra, the scarlet ibis (Fox, 1962). DISCUSS'ION

1. Melanins Our observations on the legs of the American flamingo chick are in close agreement with those of Gallct (1950), who writes of "the swollen state of the chick's feet, which have a podgy and translucent appearance. The swelling disappears after two or three days; till then the skin of the feet is a very clear pink which 8

34

D.L. Fox

turns saffron, then rose red, then grey and finally by the eighth day, almost pure black. The beak . .. undergoes the same colour-change, if somewhat lighter in tint." This sudden general melanogenesis throughout the exposed skin of the chick would appear to reflect a rapid phase of growth, involving active metabolism and redistribution of protein materials, with an accompanying elaboration or activation of the enzyme tyrosinase through cumulative effects of intermittent exposures to sunlight, and the concurrent deposition of the resulting black, lightscreening by-product melanin in the integument. As the young flamingo grows, the exposed leg-skin's blackness is very gradually replaced by a smoky or mottled black-and-pink colour-mixture until, at or near adulthood, the homogenous pink to vermilion colour is displayed over the entire surface. In the Chilean species, as has been noted, only the toes and tarsal joint are red in the adult, the rest of the leg being dark, presumably with melanin, which is seen as a bluish or greenish hue through the pale or faintly yellow, lightscattering cuticular later overlying the skin. Considering the natural habitat of flamingoes, there is manifest advantage in the presence of dark pigment in the naked leg-epidermis (i.e. the stratum corneum), which should shield the stratum germinativum from harmful sunrays before the protective horny cuticle has been elaborated. As the bird grows, the copious red carotenoids, which possess light-absorbing properties not only in the blue region of visible light but also to a lesser extent in and about the near ultra-violet portion associated with er,'thema or sunburn, mav thus afford a genuine degree of lightscreening, particularly over heel-joint and toes, where the thick scaly cuticle is not present as a screening factor. This question should be seriously im'estigated. Rapid melanogenesis and blackening of tile naked epidermis is not reported for many other vertebrates, or for more than relatively few invertebrates. Usually, melanogenesis is a gradual process, requiring some days or weeks in z'h'o, but certain insects or their larvae, elaborating the enzyme tyrosinasc, have been recorded by Gortner and others as turning from pale to very dark, melanistic colours in short periods of hours or less, e.g. cockroaches, flies, "mealxvorm" beetles, locusts, potato beetles and others (Fox, 1953). One of the striking examples of seemingly "explosive" melanogenesis in fishes occurs in both sexes, but particularly in the male, of the Tasmanian whitebait Lo,'ettia seali during gonad-ripening and immediately fl)llowing spawning, when multiple patches of dark pigmentation or even general overall blackening takes place (Blackburn, 195(I). Of common observation is the blackening of large integumentary areas in tilt" domestic ~oldfish, (?arassius auratus. 2. Effects o.f soh'ents on carotenoid colours

Brief consideration should be given here to the effects of certain solvents, their purity, and tile presence of natural oils upon the absorption spectrum of carotenoids, notably, for present purposes, the important pigment astaxanthin. Astaxanthin dissolved in petroleum ether or in methanol is yellow or, when more concentrated, orange-yellow; in pyridine a deeper orange to red-orange, and

METABOLIC FRACTIONATION, STORAGE AND DISPLAY OF CAROTENOID PIGMENTS

35

in carbon disulphide a blood-red colour. This visible deepening of colour, depending on the solvent used, is correlated with measurable increases in the wavelength of light maximally absorbed, i.e. toward the red end of the spectrum. Of particular interest in this work are the effects of natural lipids, whether present in petroleum ether extracts or serving themselves as solvents in living skin. The general effect, in either case, is to shift the absorption maximum toward the longer wavelengths. Deuel (1951) showed absorption curves for E-carotene in hexane with maxima at 448 and 476 m/~, and in Wesson oil with the respective peaks at 462-5 and 490 mF, an advancement toward the red end by some 14 m/~. It is also to be noted that the extinction coefficient (major maximum) falls from 13.75 x 10-4 in hexane to about 12.6 × 10 -q in the oil. In the present work it was observed that astaxanthin or astacene in petroleum ether systems underwent redward shifts in the centre of the maximum if appreciable quantities of accompanying colourless lipid material constituted a part of the total solvent. When pyridine was the solvent, the effect of contaminating lipids was to displace the maximum slightly backward toward the blue. The superposition of absorption curves of astacene, whether recovered from skin or from feathers, the close similarity in maximum absorption centre between astacene recovered from crayfish shell and that derived from feathers, the closely matching profiles of light-absorption exhibited by astaxanthin from crayfish shell and an astaxanthin ester from Chilean flamingo heel-skin (Fig. 4), and the curve for the free astacene derived from the latter, all provide examples of the behaviour of pyridine systems: the maximum centre lies close to its characteristic value of 490 m/~. Extracts of astaxanthin were prepared from the fresh muscle of red salmon, Oncorhynchus nerka, the so-called sock-eye or blue-back salmon from the Columbia River, and from the same species when canned (Libby's Alaska Sockeye Red Salmon). Pyridine solutions of the astaxanthin, recovered by chromatography from fresh salmon flesh, and the astacene, produced by subsequent alkaline oxidation, showed maximal absorption at 489-5 and at 493 m/~ respectively. When astaxanthin was recovered from Panulirus shell (hence involving no more than traces of contaminating lipids), its petroleum ether solution exhibited a maximum at 464 m/~ in the visible spectrum, and minor peaks in the ultra-violet at 294 and 267 m F. When astaxanthin was recovered by extraction of the canned salmon flesh, its petroleum ether solution gave a maximum at 466 and a minor peak at 365 m/~. But when the red oil from the canned product was separately dissolved in petroleum ether and transferred to triolein, the visible maximum was found to have advanced by some 12 mtz, i.e. to 478 m/z; and an extract of the canned flesh transferred to triolein exhibited its maximum at 484 m~. The triolein itself (not purified), while absorbing but little light in the visible region, exhibited a sharply rising absorption curve below 320 m#, with a high peak at about 267.5 m#. Thus, while not significantly augmenting the absorption

36

D.L. Fox

of light in the visible range, such a fatty solvent increases absorption in the near ultra-violet. The presence of natural oils may thus introduce a rising absorption toward the violet.

3. Possiblephysiological implications of skin-carotenoids It is useful to have the foregoing facts in hand when one must unavoidably work with relatively small available sources of pigments, and when it is therefore unfeasible to free the system of lipids or to crystallize the carotenoid before spectroscopic examination. But the observation may have an important physiological significance. We have already observed that in living Phoenicopterus ruber the deposits of carotenoids in naked skin exhibit not yellow or orange colours, but deep vermilion red, or, when viewed through the outer turbid cuticular layer, pink. A thin, deep-red piece of tarsal integument from a recently captured, expired American flamingo exhibited a very broad absorption spectrum, showing a small peak at 410 mix, a slight inflexion at 425 to 430 mix, followed by a long, gently sloping plateau from 445 to about 560 m/~ preceding a downward trend to 700 m/z (Fig. 16). Attention is again called to the earlier observation of an absorption band at 490 mt~ for native blood plasma, and to the maximum at 484 mix of astaxanthin in triolein. But perhaps of greater significance is the absorption in the near ultra-violet, e.g. from 295 up to 320 m/~, the range causing solar erythema (Blum, 1955), by astaxanthin and notably by that carotenoid in natural oily fats. Moreover, the skin exhibited high absorption of light well down into the violet (375 m/z). This may have been attributable partly to general scattering, as may the absorption in the red end of the spectrum, notwithstanding the use of an opalglass plate to minimize such effects. The denaturation of the skin tissue by alcohol, acetone or other extracting fluids would have nullified any usefulness of its reexamination spectroscopically when thus leached of pigment. Flamingo chicks with naked clear skin over their exposed legs should suffer local burns from the blazing sunlight of their native tropical habitat unless provided with chemical means for shielding the delicate tissues from the injurious portion of the incident spectrum. This the young bird seemingly achieves as soon as its shanks are no longer to be shaded by its own downy body or by the parents. For we have seen that, at the time that the nestling begins its wanderings, and considerably before anything in the nature of a light-filtering cuticle has covered the surface, heavv deposits of melanin suddenly appear in dermis and epidermis (Fig. 9). This striking event is in line with the finding, in the integument of other animals, that not only may pigment migration toward the upper layers of skin begin some 24 hr after an initial exposure to ultra-violet rays, but actual pigment darkening, i.e. dark coloration of melanin precursors, is effected within hours or minutes by exposure to light in the range from about 300 to 420 mix, with a broad action-maximum at about 320 mix (Blum, 1955). As the juvenile bird approaches adulthood, a reversal of integumentary melanization occurs as the dark colour gives way to pink or red. The carotenoids

METABOLIC

FRACTIONATION,

STORAGE

AND

DISPLAY

OF CAROTENOID

PIGMENTS

37

responsible for the new coiour perhaps serve little or no ultra-violet light-screening role in those portions of the featherless skin covering the long leg-bones and now overlain ~by a heavy, light-scattering cuticle (although it is recalled that the Chilean species retains its melanin in those parts, giving them greenish or bluish shades), but may very conceivably serve such a useful function at the tarsal joint and over the toes, where the cuticle would be far thinner, and where both species, especially the Chilean birds, exhibit very rich carotenoid deposits. Blum (1955), referring to mammalian skin, suggests that, while thickening of the corneum probably constitutes a more effective filter against harmful sunrays than does melanin, this pigment may still play some part in the screening process. 07

m.~'o N~ o °~

o do5

o° %°~o.

Od O,

"°. :

. . . . . . . . .

.

.

L ".

°~'°.°';

FIG. 16. Absorption spectrum of fresh shank-skin from P. tuber (opal-glass filter). This must hold true likewise in the skin of many birds, notably in those possessing a heavy scale covering the exposed portions of the legs and feet. In the American flamingo chick, however, wherein the exposed leg-skin is without a scaly protein later, the rich deposits of melanin microspheroids in the very surface of the epidermis may well serve to protect the Malpighian layer from erythemal rays. The extremely thin skin covering the adult's shank must receive ample protection from the overlying scale. The gradual disappearance of melanin from the exposed skin of the growing bird, giving way to carotenoid deposition in the relatively tough skin covering the heel-joint and the upper surfaces of toes and web, may also be accompanied by a thickening of the protective corneum, which, in non-jointed parts of the leg, elaborates the envelope of dead, horny keratin. Carotenoids dissolved in fatty oils should be capable of effectively screening out injurious sunrays in the critical region between 295 and 320 m#. This whole question calls urgently for a careful experimental investigation, perhaps based upon withholding carotenoid-rich food from young, growing flamingoes and exposing them to erythemal doses of light. Much, meanwhile, remains to be learned about the sites and mechanisms of carotenoid fractionation in adult flamingoes. It appears that these birds, receiving copious supplements of animal or plant carotenoids in their food, are able to elaborate, from such precursors, many new carotenoids, including astaxanthin, canthaxanthin, some other unique xanthophyUs, and a few unusual epiphasic compounds, some of them resembling certain dehydrocarotenes.

38

D . L . Fox

W h i l e t h e i n t e s t i n a l flora d e m o n s t r a b l y c o n t a i n e d d e h y d r o g e n a s e s c a p a b l e o f affecting c a r o t e n o i d m a t e r i a l (e.g. astacene) inter alia, a n d while t h e d i e t a r y c a r o t e n o i d s , l a c k i n g in t h e faeces, m u s t be largely d e s t r o y e d in t h e gut, s o m e q u e s t i o n s await answers, e.g. w h e r e are c a n t h a x a n t h i n and, on an a s t a x a n t h i n - f r e e diet, a s t a x a n t h i n itself e l a b o r a t e d ? Both o f these are f o u n d in s u b s t a n t i a l q u a n t i t i e s in t h e feathers of P. ruber, a n d a s t a x a n t h i n in t h e leg-skin of b o t h P. ruber a n d P. chilensis as well. C a n t h a x a n t h i n , not i d e n t i f i e d in the skin, a p p e a r e d in t h e egg volk of P. ruber. But n e i t h e r of these k e t o c a r o t e n o i d s c o u l d be r e c o g n i z e d in the c a r o t e n o i d - r i c h b l o o d s t r e a m , nor in any of the o t h c r s o m a t i c tissues, d e s p i t e the a s t a x a n t h i n in the diet. I t t h u s w o u l d a p p e a r p o s s i b l e e i t h e r t h a t b o t h m a y be e l a b o r a t e d hy t h e g u t flora, and f o r t h w i t h p a s s e d over to t h e blood, t h e r e i n to be t r a n s p o r t e d i m m e d i a t e l y a n d in very m i n u t e , u n d e t e c t a b l e q u a n t i t i e s , to t h e i r r e s p e c t i v e sites of d e p o s i t i o n , or t h a t o t h e r c a r o t e n o i d p r o d u c t s , e l a b o r a t e d in the l u m e n or m u c o s a of t h e gut a n d p r e s e n t in the b l o o d , are f u r t h e r m o d i f i e d bv cellular s e c r e t i o n s at the res p e c t i v e sites, e.g. to a s t a x a n t h i n n e a r the base of t h e g r o w i n g l e a t h e r a n d in the leg-skin, a n d to c a n t h a x a n t h i n as well in the living f e a t h e r - p a r t s and in the y o l k s e c r e t i n g gland.

Acknozcledgements---It is a privilege to record my indebtedness to a nunzber of associates whose help and encouragement were of inestimable value to this research. Without the sustained and ever-willing cry-operation of officers and assistants at the San I)iego Zoo, the work could not have been accomplished. Accordingly, ] ~we a lasting debt of gratitude to Dr. Charles R. Schrocder, l)irector; Mr. K. C. l,int, Curator of Birds, and his staff; Mr. Merl Moody, manager of the warehouse; Dr. Anthony Sylstra, sometime Veterinarian, Miss Alice Hansen and Mr. B. Sheridan, laboratory technicians, and Dr. Wcrner P. Heuschele, fi~rmerly Veterinarian and Manager of the Zoo's llospital and Biological Research Institute. [ am indebted to Mr. Attain Schmidt, Preparator at the San Diego Museum of Natural History, and to Mr. Sheridan for fresh tissues from flamingo chicks; to Miss Thea Schultze, microtechnician at S. I. O., for embedding and sectioning heel-skin from the chick; to Dr. T. J. \Valker at S. I. O. for kindly preparing unstained mounted sections of feather barbs ; to Dr. Daniel .Mazia and l)r. ttoward Bern in the Department of Zoology at the University of California, Berkeley, for nlicrophotographs of the chick's skin-sections; and to I)r. Richard Y. :'klorita for examining the dehydrogenase activities of flamingo gut-microflora. Among the earlier and present staff in my division (Biochemistry) to whom c~me nay thanks are Dr. Arthur I,. Kelly, Dr. Eugene F. Corcoran, l)r. Wheeler J. North, Mr. James S. Kittredge, Mr. Michael Pilson, and, in the Botany Division, Mrs. Nao Belser, fl)r varigms kinds of technical help. l am grateful tc, my colleague Professor Francis T. I laxo and to l)r. S. K. lZon for reviewing the manuscript and for helpful suggestions; to Prc~fessor 1,. Zechmeister for it sample of pure canthaxanthin, as a referencepigment, and to Mr. \V. (;. Conway of the New York Zoo and ])r. Holger Poulsen of the Copenhagen Zoo for helpful infi)rmation and for valuable feather specimens. [ wish to dedicate this paper to my late son Stephen J. P. Fox (1935---1954), university student and summer-time assistant keeper in the bird-yard of the San Diego Zoo, notably for his abiding interest and enthusiasm, his early dedicated part in the care and feeding of the flamingo flock, and the collection therefrom of research materials for analysis.

METABOLIC FRACTIONATION, STORAGE AND DISPLAY OF CAROTENOID PIGMENTS

39

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McCANN C. (1939) The flamingo (Phoenicopterus ruber antiquorum rI'emm.). J. Bombay Nat. Hist. Soc. 41, 12-38. POULSEN H. (1960) Colour feeding of flamingoes. Avicult. Mag. 66, 48-51. RIDLEY M. W. (1954) Observations on the diet of flamingoes. J. Bombay Nat. Hist. Soc.

52, 5-7. RIDLEY M. W., Moss B. L. & PERCY R. C. (1955) The food of flamingoes in Kenya Colony. J. E. Afric. Ug. Nat. Hist. Soc. 22, 147-158. STRAIN H. H. (1942) The occurrence and interconversion of various fucoxanthins. J. Amer. Chem. Soc. 64, 1235. V6Lmm O. (1950) Astaxanthin als Federpigment. Naturwiss. 13, 309- 310. V6L~R O. (1954a) Die Isolierung yon Astaxanthin aus Federn. Naturrdss. 17, 405. V6LKER O. (1954b) Die Natur und die Herkunft roter Lipochrome in der Klasse der V6gel. Bet. Oberhess. Ges. Nat- u. Heilk. 27, 58-66. V6LKER O. (1955a) Die schwartzroten und violetten lipochromatischen Federfarben tier Cotingiden. Naturwiss. 22, 612-613. V6LKF.a O. (1955b) Die Isolierung von Astaxanthin aus den Federn des Rotbauchwtirgers Laniarius atrococcineus, ft. Orn. Lpz. 96, 50-53. V6LKER O. (1958) Die Rotf~irbung der Flamingos (Phoenicopterus) im Freileben und in der Gefangenschaft. J. Orn. Lpz. 99, 209-217. WALD G. & ZUSSMA~,"H. (1937) Carotenoids of the chicken retina. Nature Lond. 140, 197. WALt) G. & ZUSSMA.~ H. (1938) Carotenoids of the chicken retina. )'. Biol. Chem. 122, 449--460. ZECHMEISTER L. (1960) Cis-trans isomeric carotenoid pigments. Fortschr. Chem. org. Naturst. 18, 223-349.