A number of recent studies have examined the role of antioxidant protection in avian ecology and physiology (e.g. Hõrak et al. 2006; Tummeleht et al. 2006; Alonso-Alvarez et al. 2007; Isaksson et al. 2007). Antioxidants are valuable both for protection against free radical damage – considered to be an important mechanism underlying aging – and for proper functioning of the immune system. However, results have not always been straightforward. Depending on species and ecology, higher antioxidant capacity can be indicative of animals in good or poor condition (Costantini & Dell’Omo 2006; Costantini, Cardinale & Carere 2007), and can rise or fall in response to stress (Cohen, Klasing & Ricklefs 2007). At the same time, one type of antioxidant – carotenoids – has repeatedly been shown to be important for good health and for sexual signalling in a number of species (e.g. Blount et al. 2003; McGraw & Ardia 2003), though it is not clear that these benefits come from the role of carotenoids as antioxidants. Supplementation of the common carotenoid lutein in chickens (Gallus domesticus) decreased overall antioxidant capacity, though vitamin E supplementation increased antioxidant capacity (Cohen et al. 2007). In contrast, in zebra finches (Taeniopygia guttata), carotenoid supplementation increased overall antioxidant protection (Alonso-Alvarez et al. 2004). Intermediate but not high levels of vitamin E supplementation improved body condition in nestling barn swallows (Hirundo rustica) (de Ayala, Martinelli & Saino 2006).
This range of conflicting results is particularly difficult to interpret given our poor understanding of the interactions among components of antioxidant systems and of complexity in physiological systems more generally. The growing use of markers of overall antioxidant capacity implies a conception in the literature of antioxidants as a unified system that can be summarized by a single variable. If antioxidant function is modulated as a coherent whole via regulation within individuals or selection across individuals or species, we should expect strong correlations among different types of antioxidants at those respective levels, either positive (suggesting synergism) or negative (suggesting compensatory effects). However, it is not clear that overall antioxidant function in circulating systems is more than the sum of a number of largely independent processes affecting the levels of individual antioxidant types, potentially neither regulated nor subject to selection with regard to antioxidant function (Hartley & Kennedy 2004). Even to the extent that each antioxidant type is regulated for its antioxidant function, the functions may be type-specific enough that there is relatively little signal in overall levels. For example, although carotenoid levels are quite low relative to overall antioxidant levels, they are considered to play important free radical scavenging roles in membranes (Young & Lowe 2001). Carotenoids are now considered not to contribute much too overall antioxidant status (Isaksson et al. 2007; Costantini & Møller 2008).
For these reasons, the question of how to measure antioxidants is related to a fundamental understanding of the physiological roles of different antioxidant types and the degree of their integration into a single coherent system. These questions can be addressed for at least three different ecological levels: across species, reflecting regulation over evolutionary time; within species across individuals, reflecting covariation based on genetics, diet or condition; and within individuals over time. We have observed strong covariation among antioxidants within individuals in response to capture stress, and describe this relationship elsewhere (Cohen 2007). Here, we focus on inter- and intraspecific variation.
Antioxidant systems involve both enzymes and micromolecules and vary across tissues. Enzymatic antioxidants are particularly important in mitochondria at the site of most free radical production (e.g. Van Remmen et al. 2003). Here we focus on circulating micromolecular systems, known to affect both immune function and oxidative damage (Konjufca et al. 2004; McGraw & Ardia 2004; Niki 2004). Most recent work on antioxidants in an ecological context has focused on micromolecular antioxidants in birds. There is clear evidence that for some antioxidants dietary availability is a critical factor. Vitamin E and carotenoids cannot be produced endogenously, though some types of carotenoids can be modified to others, depending on the presence of the appropriate enzymes (Surai 2002). Others, such as vitamin C and uric acid, can be produced endogenously at least in some birds, and additionally uric acid is the main by-product of amino acid metabolism in birds, and its levels thus may reflect protein intake or regulation of nitrogen excretion as much as antioxidant function (Wright 1995). In many bird species, uric acid is also by far the most abundant of the circulating micromolecular antioxidants (as opposed to the enzymatic antioxidants, which function primarily in mitochondria), and uric acid levels have been shown to correlate well with the Trolox-equivalent antioxidant capacity measure (TEAC, Cohen et al. 2007; Hõrak et al. 2007). Whether or not there are particularly strong correlations within groups of antioxidants such as lipid- vs. water-soluble molecules or those with exogenous vs. endogenous sources will clarify the functional relevance of these classes.
Here, we studied circulating antioxidants in more than 900 individuals from 99 wild bird species. For almost all individuals, we quantified TEAC, uric acid and non-uric acid (residual) antioxidant capacity. For 428 individuals we also measured vitamin E and carotenoid levels. Four different types of carotenoids, including both carotenes and xanthophylls, were found in enough species to be analyzed individually, in addition to total carotenoid concentration and number of carotenoid types present. At least in vitro, carotenes generally are more effective antioxidants than xanthophylls due to the absence of a hydroxyl group on the β-ring (Miller et al. 1996).
We examined correlations among these antioxidant measures at the interspecific level and at the intraspecific level for 30 different species for which we had sufficient sample size (n ≥ 5) and confirmed these analyses with multi-level models assessing the heterogeneity of correlations across species and the partitioning of variance between the individual and species levels. We were interested in understanding the correlation structure among different antioxidant types, its variation across species, and how well a summary measure such as TEAC would capture this variation. A correlational study such as this is not intended to shed light on causal links between these antioxidants (e.g. whether they are co-regulated, associated because of diet, etc.; see Discussion), but is an important starting point from which experiments on specific antioxidants can be designed and interpreted. For example, an experiment showing that immune stimulation causes an increase in antioxidant capacity and a decrease in carotenoid levels has different implications depending on whether antioxidant capacity and carotenoids are known to consistently covary on a broader scale. Heterogeneity in the correlations across different species would suggest that the associations, rather than being strictly physiologically constrained, either evolve themselves or are determined by the evolution of other traits such as diet and habitat. Most importantly, all these patterns can serve as a proxy for understanding complexity in physiological systems more generally.