SHORT COMMUNICATIONS 587 The Condor 105:587±592 q The Cooper Ornithological Society 2003 CAROTENOID PIGMENTS IN A MUTANT CARDINAL: IMPLICATIONS FOR THE GENETIC AND ENZYMATIC CONTROL MECHANISMS OF CAROTENOID METABOLISM IN BIRDS KEVIN J. MCGRAW1,2,4,GEOFFREY E. HILL2 AND ROBERT S. PARKER3 1Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853 2Department of Biological Sciences, 331 Funchess Hall, Auburn University, Auburn, AL 36849 3Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853 Abstract. Birds that use carotenoids to color their los pigmentos carotenoides presentes en las plumas de feathers must ultimately obtain these pigments from un cardenal Cardinalis cardinalis con coloracioÂn ama- the diet, but they are also capable of metabolically rilla aberrante para determinar coÂmo se podrõÂan haber transforming dietary carotenoids into alternate forms alterado las rutas metaboÂlicas. Los cardenales norma- that they use as plumage colorants. The genetic and les de color rojo despliegan cuatro keto-carotenoides enzymatic control mechanisms underlying carotenoid primarios en el plumaje, los cuales son derivados en- metabolism are poorly understood. We investigated ca- doÂgenamente a partir de una serie de carotenoides co- rotenoid pigments present in the feathers of an aber- munes en la dieta. Encontramos que las plumas ama- rantly colored yellow Northern Cardinal (Cardinalis rillas del mutante carecõÂan de los cuatro pigmentos ro- cardinalis) to determine how metabolic pathways may jos tõÂpicos, y contenõÂan en cambio un carotenoide die- have been altered. Normal red cardinals display four tario amarillo (luteõÂna) y tres derivados metaboÂlicos primary keto-carotenoids in plumage that are endoge- amarillos (39-dehidro luteõÂna y xanto®las de canario A nously derived from a series of common food carot- y B). Debido a que los metabolitos amarillos parecen enoids. We found that the yellow feathers of this mu- ser fabricados mediante un proceso metaboÂlico dife- tant lacked all four of the typical red pigments, and rente al de las formas rojas usuales (deshidrogenacioÂn instead contained a yellow dietary carotenoid (lutein) y oxidacioÂn en las posiciones C-4 o -49, respectiva- and three yellow metabolic derivatives (39-dehydrolu- mente), parece que esta mutacioÂn geneÂtica no altero tein and canary-xanthophylls A and B). Because yel- ampliamente toda la actividad metaboÂlica. En cambio, low metabolites appear to be manufactured via a dif- parece haber inhabilitado soÂlo una clase de reacciones ferent metabolic process (dehydrogenation) than the metaboÂlicas y sus enzimas asociadas. usual red forms (oxidation at the C-4 or -49 positions), it seems that this genetic mutation did not broadly dis- rupt all metabolic activity, but speci®cally impaired For nearly a century, biologists have known that ca- only one class of metabolic reactions and its associated rotenoid pigments cannot be synthesized de novo by enzymes. vertebrates (Palmer 1922). Instead, carotenoids must Key words: Cardinalis cardinalis, carotenoids, me- be obtained in the diet to serve various physiological tabolism, mutation, Northern Cardinal. functions or to be used in integumentary color displays (Olson and Owens 1998, Hill 1999, Mùller et al. 2000). Animals can, however, metabolically alter in- Pigmentos Carotenoides en un Cardenal gested pigments (Davies et al. 1970, Brush 1990, Mutante: Implicaciones para los Mecanismos Schiedt 1990). Birds, for example, consume a variety GeneÂticos y EnzimaÂticos de Control del of dietary carotenoids, including the hydroxycarote- Metabolismo de Carotenoides en las Aves noids lutein and zeaxanthin, but often deposit into feathers and bare parts more oxidized forms, such as Resumen. Las aves que utilizan pigmentos caro- the canary-xanthophylls that are yellow in color or red tenoides para dar color a sus plumas deben obtenerlos keto-carotenoids like astaxanthin (Stradi 1998). de la dieta, pero tambieÂn son capaces de transformar Despite recent interest in the proximate and ultimate metaboÂlicamente los carotenoides dietarios en formas bases for variation in carotenoid pigmentation in birds alternativas que utilizan para colorear las plumas. Los (Hill 2002), the particular enzymatic and genetic mecanismos geneÂticos y enzimaÂticos de control invo- mechanisms controlling avian carotenoid metabolism lucrados en el metabolismo de los carotenoides no han remain essentially undescribed. Only in domestic sido bien establecidos. En este estudio investigamos chickens (Gallus domesticus) have dietary carotenoids been radiolabeled in vivo to determine the metabolic fate of ingested precursor pigments in birds (Schiedt Manuscript received 10 January 2003; accepted 14 1990, 1998). Moreover, only in studies of b-carotene April 2003. as a vitamin-A precursor have enzymes been identi®ed 4 E-mail: [email protected] that catalyze carotenoid metabolism in animals (Wyss 588 SHORT COMMUNICATIONS FIGURE 1. Reference photograph of the mutant yellow cardinal, placed next to a museum specimen of a wild- type red cardinal. et al. 2000), and this central-cleavage process is much dinal in North America. Even when fed carotenoid- different from the end-ring oxidation reactions that are deprived diets in captivity during molt, male cardinals more common in birds (Stradi 1998, Stradi et al. still grow a pale red plumage, manufacturing the typ- 2001). Ultimately, to better understand how and why ical suite of red keto-carotenoids from the low con- birds complete these pigment transformations, more centration of available dietary carotenoids (McGraw et consideration should be given to the biochemical and al. 2001). Thus, it seems that a de®ciency of dietary genetic systems that regulate the enzyme(s) that me- carotenoids should not have induced this individual to tabolize dietary carotenoids. grow yellow feathers, but instead that this bird may Studies of mutant individuals that fail to display the have lost its ability to metabolize carotenoids from the species-typical plumage-color pattern (e.g., VoÈlker diet through the disruption of genetically determined 1964, Hudon 1997) may provide valuable insights into metabolic pathways. We obtained a sample of yellow the genetic and enzymatic systems of carotenoid me- contour feathers from this specimen and used high- tabolism in colorful birds. In this study, we compared performance liquid chromatography (HPLC) to deter- the carotenoid-pigment pro®le in the plumage of an mine its plumage-carotenoid composition so that it aberrantly colored male Northern Cardinal (Cardinalis could be compared to the pigment pro®le of wild male cardinalis) to that of wild-type individuals to under- cardinals (McGraw et al. 2001). stand the degree to which changes in metabolic path- ways generated this plumage-color variant. Wild-type METHODS male Northern Cardinals are bright red, and their red In April 2001, we obtained 6 pigmented contour feath- feather carotenoids are synthesized endogenously from ers from this yellow male Northern Cardinal from the the yellow and orange precursor pigments they obtain LSU Museum of Natural Science. These feathers were from the diet (McGraw et al. 2001). However, in July divided into two groups of three, and the carotenoids 1989, an adult male cardinal with uniformly yellow were extracted from each using two different methods: plumage was collected in Baton Rouge, Louisiana, by a thermochemical procedure (Hudon and Brush 1992), the Museum of Natural Science at Louisiana State Uni- and a mechanical procedure (Stradi, Celentano, and versity (museum specimen # LSUMZ 139415; Fig. 1). Nava 1995). Prior to each procedure, we separately To our knowledge, this is the lone reported yellow car- washed the feathers in 5 mL ethanol and 5 mL hexane SHORT COMMUNICATIONS 589 for 30 min, blotted them dry, and trimmed off the ca- rotenoid-pigmented barbules for analysis. For the ther- mochemical method, we added 1 mL acidi®ed pyridine to the barbules in a 9-mL glass tube, ®lled the head- space with argon, and held the solution at 958C for 3 hr. The tube was then cooled to room temperature and we extracted the lipids by adding 1 mL distilled water and 5 mL hexane:tert-butyl methyl ether (1:1) and shaking vigorously for 2 min. For the mechanical method, we ground pigmented barbules for 15 min at 30 Hz using a Retscht MM200 mixer mill (Retsch Inc., Irvine, California), equipped with a zirconia grinding jar and balls (and Te¯ont O-ring) and in the presence of 3 mL methanol. At this point in both pro- cedures, we centrifuged the resulting mixtures for 5 min at 3000 rpm, transferred the supernatant to a clean tube, and evaporated the solvent to dryness under a stream of nitrogen. The pigment residue was resuspen- ded in 200 mL of HPLC mobile phase (methanol:ace- tonitrile:chloroform, 46:46:8) prior to analysis. We injected 50 mL of each sample into a Watersy 717plus Autosampler HPLC (Millipore Corp., Bed- ford, Massachusetts) ®tted with a Develosil RPA- queous RP-30 column (250 3 4.6 mm; Nomura Chem- ical Co. Ltd., Aichi, Japan) and an Eppendorf TC-50 column heater (Hamburg, Germany) set at 278C. We used an isocratic system (Hewlett-Packard 1050 Series FIGURE 2. Three-dimensional HPLC chromatogram Isocratic Pump) at a constant ¯ow rate of 0.6 mL min21 of the carotenoid pigments present in the mutant yel- for 60 min to allow suf®cient time for both xantho- low plumage of a male cardinal. Lutein and 39-dehy- phylls and carotenes to elute if they were present. Data drolutein were the major plumage carotenoids, com- were collected from 250±600 nm using a Watersy 996 prising 90% of total eluents, whereas canary-xantho- photodiode array detector (Waters Corporation, Mil- phylls A and B were minor constituents. Total carot- ford, Massachusetts). We identi®ed pigments by com- enoid concentration in mutant feathers was 0.73 mg paring their respective retention times and absorbance g21, which falls within the range of variation observed maxima to those of authentic reference carotenoids run in wild red cardinals and other carotenoid-pigmented as external standards. The relative abundance of these songbirds (KJM, unpubl.