Identifying anatomical sites of carotenoid metabolism in birds.

Carotenoid metabolism has long interested plant and animalbiochemists (Goodwin 1986; Lu and Li 2008). Identifyingtissue sites and enzymes responsible for carotenoid trans-formations (e.g., β-carotene to vitamin A) has beenchallenging. Colorful birds have recently become a modelfor studying carotenoid nutrition and metabolism, in thecontext of sexual selection and honest signaling (McGraw2006). In a recent paper published in Naturwissenschaften,del Val et al. (2009) described carotenoid profiles in tissuesof male common crossbills (Loxia curvirostra), with theaim of localizing metabolic site(s) for a ketocarotenoidpigment—3-hydroxy-echinenone (3HE)—present in redfeathers. They found 3HE in blood and liver, unlikeprevious studies of colorful songbirds where metabolizedintegumentary carotenoids were found only at peripheraltissues (e.g., beak, legs; McGraw 2004). Thus, the authorsconcluded that the liver was the site of ketocarotenoidsynthesis in crossbills. Here, I outline the limitations ofsuch descriptive pigment analyses, when carotenoid typesare present in various tissues, and discuss fruitful directionsfor future lines of research in this area.Because carotenoids are dietary in origin in animals butcan be modified in body tissues enzymatically, careful foodand physiological studies allow for accurate identificationof pigment origins and transformations. Traditional rigor-ous methods for identifying carotenoid metabolic sites,processes, and products in animals include direct enzymeidentification (Wyss 2004), isotope labeling of precursors(Burri and Clifford 2004), and by inference from ex vivochemical reactions (Khachik et al. 1998) or where caroten-oid types exist in no other tissue type (McGraw 2004).del Val et al. (2009) undertook none of these types ofinvestigation. The first step in such research is to rule out adietary source to the pigment, but the authors did not studyfood carotenoids in crossbills; they sampled only liver,skin, and feathers from accidentally field-killed animals anddrew blood from molting birds. While red carotenoids arenot currently thought to be common in diets of herbivorousland birds (e.g., rubixanthin in rose hips, rhodoxanthin inTaxus berries), this is a key assumption to biochemicallyvalidate for any species, given the paucity of informationon avian food carotenoids.del Val et al. (2009) based their assertion that 3HE wasmetabolized in the liver of crossbills on the observation that3HE was the most proportionally concentrated (and mostvariably occurring) carotenoid in liver. However, data onother types of body carotenoids were not presented in thispaper, the molt status of dead birds was not given, and withsmall sample sizes (n=7) no statistics were performed.Thus, we have no context for understanding which and howcertain carotenoid pigments occurred in different crossbilltissues. Follicles actually contained proportionally more3HE than liver (61% vs. 35% of total carotenoids,respectively), and while the authors argue that this isexpected (given that “maturing follicles would just accu-mulate this red derivative until the pigment would beincorporated finally into growing feathers during moult”;p. xxx), this makes the non-null assumption that folliclespreferentially retain 3HE over other circulating carotenoids.Even if we assume that 3HE is not dietary in origin,alternate tissues with carotenoidmetabolismcapabilitieswerenot studied. The oxygenase enzyme that cleaves β-carotene