A quantitative comparison of the commonly used methods for extracting carotenoids from avian plasma
- 199 Downloads
Interest in animal carotenoids, especially in birds, has exploded in recent years, and so too have the methods employed to investigate the nature and function of these pigments. Perhaps the most easily and commonly performed procedure in this work has been the determination of carotenoid concentration from avian plasma. Over the past 20 years of research on avian carotenoids, numerous methods have been used to extract carotenoids from bird plasma, all of which have differed in several important parameters (e.g., number and types of solvents used, degree of mixing/centrifugation). However, to date, no study has systematically compared these methods to determine if any of them are more effective than others for recovering any or all types of carotenoids present. We undertook such an investigation on plasma samples from two bird species (house finch, Carpodacus mexicanus, and mallard, Anas platyrhynchos) using five of the most commonly employed methods for extracting carotenoids from avian plasma: (1) acetone-only, (2) methanol-only, (3) ethanol-only, (4) ethanol + hexane, and (5) ethanol + tert butyl methyl ether. We also manipulated the amount of time that extracts were centrifuged, which has varied tremendously in previous studies, to evaluate its importance on carotenoid recovery. We found that all methods equally recovered the polar xanthophylls (lutein and zeaxanthin), but that the methanol-only procedure poorly recovered non-polar carotenoids (less β-carotene in both species and less β-cryptoxanthin in house finches) compared to the other methods. These results suggest that the data accumulated to date on xanthophyll plasma carotenoids in birds should be comparable across studies and species despite the different chemical extraction methods used. However, care should be taken to use relatively strong organic solvents for fully recovering non-polar carotenoids. We also found no effect of centrifugation duration (1 vs. 10 min at 10,000 rpm) on carotenoid recoveries, demonstrating that researchers can save considerable time by centrifuging for a much shorter time period than is typically used.
KeywordsCarotenoid pigments Ethanol House finch HPLC Lutein Mallard Methanol Zeaxanthin
We thank S. Quinn for assistance in capturing ducklings as well as two anonymous referees for providing helpful comments on the manuscript. Financial support for this study was provided by the School of Life Sciences and College of Liberal Arts and Sciences at Arizona State University. Birds from both species were captured and sampled under university (protocol nos. 05-764R and 07-910R), state (SP797514), and federal (MB088806-0) permits.
- Casagrande S, Costantini D, Fanfani A, Tagliavini J, Dell’Omo G (2007) Patterns of serum carotenoid accumulation and skin colour variation in kestrel nestlings in relation to breeding conditions and different terms of carotenoid supplementation. J Comp Physiol B 177:237–245PubMedCrossRefGoogle Scholar
- McGraw KJ (2006a) The mechanics of carotenoid coloration in birds. In: Hill GE, McGraw KJ (eds) Bird coloration. I. Mechanisms and measurements. Harvard University Press, Cambridge, MA, pp 177–242Google Scholar
- McGraw KJ, Ardia DR (2005) Sex differences in carotenoid status and immune performance in zebra finches. Evol Ecol Res 7:251–262Google Scholar
- Negro JJ, Tella JL, Hiraldo F, Bortolotti GR, Prieto P (2001) Sex- and age-related variation in plasma carotenoids despite a constant diet in the red-legged partridge (Alectoris rufa). Ardea 89:275–280Google Scholar
- Staciewicz-Sapuntakis M, Bowen P, Kirkendall J, Burgess M (1987) Simultaneous determination of serum retinol and various carotenoids: their distribution in middle-aged men and women. J Micronutr Anal 3:27–45Google Scholar
- Stradi R, Celentano G, Rossi E, Rovati G, Pastore M (1995) Carotenoids in bird plumage—I. The carotenoid pattern in a series of Palearctic Carduelinae. Comp Biochem Physiol 110B:131–143Google Scholar
- Tummeleht L, Magi M, Kilgas P, Mand R, Horak P (2006) Antioxidant protection and plasma carotenoids of incubating great tits (Parus major L.) in relation to health state and breeding conditions. Comp Biochem Physiol C 144:166–172Google Scholar
- Wilson WO (1956) Identifying non-laying chicken hens. Poult Sci 35:226–227Google Scholar