Plasma Non-enzymatic Antioxidant Capacity Research Articles

A blood-based multi-biomarker approach reveals different physiological responses of common kestrels to contrasting environments

The increase of urbanization and agricultural activities is causing a dramatic reduction of natural environments. As a consequence, animals need to physiologically adjust to these novel environments, in order to exploit them for foraging and breeding. The aim of this work was to compare the physiological status among nestling common kestrels (Falco tinnunculus) that were raised in nest-boxes located in more natural, rural, or urban areas in a landscape with a mosaic of land uses around Rome in Central Italy. A blood-based multi-biomarker approach was applied to evaluate physiological responses at multiple levels, including antioxidant concentrations, immunological functions, genotoxicity, and neurotoxicity. We found lower concentrations of glutathione and GSH:GSSG ratio values and higher proportions of monocytes in urban birds compared to the other areas. We also found higher DNA damage in rural compared to urban and natural krestels and inhibition of butyrylcholinesterase activity in urban and natural birds compared to rural area. Finally, we found similar values among study areas for respiratory burst, complement system, bactericidal capacity, and plasma non-enzymatic antioxidant capacity. These results suggest that (i) city life does not necessarily cause physiological alterations in kestrels compared to life in other habitats, and (ii) environmental pressures are likely to differ in typology and intensity across habitats requiring specific responses that a multi-biomarker approach can help to detect. Further studies are needed to assess which factors are responsible for the physiological differences among city, rural, and natural birds, and whether these differences are consistent across time and space.

Strenuous swimming raises blood non-enzymatic antioxidant capacity in rats.

The non-enzymatic antioxidant system protects blood components from oxidative damage and/or injury. Herein, plasma non-enzymatic antioxidant capacity after acute strenuous swimming exercise (Exe) and exercise until exhaustion (Exh) was measured in rats. The experiments were carried out in never exposed (Nex) and pre-exposed (Pex) groups. The Nex group did not undergo any previous training before the acute strenuous swimming test and the Pex group was submitted to daily swimming for 10 min in the first week and 15 min per day in the second week before testing. Plasma glucose, lactate, and pyruvate were measured and plasma total protein sulfhydryl groups (thiol), trolox equivalent antioxidant capacity (TEAC), ferric reducing ability of plasma (FRAP), and total radical-trapping antioxidant parameter (TRAP) levels were evaluated. There were marked increases in plasma lactate concentrations (Nex-Control 1.31±0.20 vs NexExe 4.16±0.39 vs NexExh 7.19±0.67) and in thiol (Nex-Control 271.9±5.6 vs NexExh 314.7±5.7), TEAC (Nex-Control 786.4±60.2 vs NexExh 1027.7±58.2), FRAP (Nex-Control 309.2±17.7 vs NexExh 413.4±24.3), and TRAP (Nex-Control 0.50±0.15 vs NexExh 2.6±0.32) levels after acute swimming and/or exhaustion. Also, there were increased plasma lactate concentrations (Pex-Control 1.39±0.15 vs PexExe 5.22±0.91 vs PexExh 10.07±0.49), thiol (Pex-Control 252.9±8.2 vs PexExh 284.6±6.7), FRAP (Pex-Control 296.5±15.4 vs PexExh 445.7±45.6), and TRAP (Pex-Control 1.8±0.1 vs PexExh 4.6±0.2) levels after acute swimming and/or exhaustion. Lactate showed the highest percent of elevation in the Nex and Pex groups. In conclusion, plasma lactate may contribute to plasma antioxidant defenses, and the TRAP assay is the most sensitive assay for assessing plasma non-antioxidant capacity after strenuous exercise.

Diet, antioxidants and oxidative status in pygoscelid penguins

Ecologically similar marine species inhabiting the same areas compete for food resources. Such competition is reduced by resource-partitioning strategies that may affect physiology. For instance, diet and feeding strategies may affect the antioxidant defences or the production of reactive oxygen species. Oxidative stress is defined as the imbalance between pro-oxidants and antioxidant defences. If such an imbalance favours the former, this can lead to oxidative damage, and oxidative stress increases. However, to our knowledge, how free-ranging animals adjust their oxidative status in relation to their foraging habitats, diet and dietary antioxidants has not yet been studied. Penguins are an interesting biological model for such a comparison because their diet, based on krill, fish and/or cephalopods, presents strong variation in dietary antioxidant content. We therefore examined trophic level (δ15N), foraging habitat (δ13C), dietary antioxidants (retinol, α-tocopherol and astaxanthin) and oxidative status (plasma non-enzymatic antioxidant capacity and oxidative damage) in pygoscelid penguins (chinstrap Pygoscelis antarcticus, gentoo P. papua and Adélie P. adeliae) breeding in Antarctica. We found interspecific differences in all variables analysed except α-tocopherol. Gentoo penguins exploited more cephalopods and fish in coastal and benthic habitats, Adélies showed an intermediate position, whereas chinstraps foraged more on krill and fish in pelagic waters. Dietary antioxidant levels showed specific patterns resulting in relationships with prey items. However, we did not find any clear relationships between dietary antioxidants and species-specific antioxidant capacity, suggesting the importance of endogenously produced antioxidants. Oxidative status appeared to be differently related to foraging strategy and antioxidant capacity in each species.

Determinants of defence strategies of a hibernating European bat species towards the fungal pathogen Pseudogymnoascus destructans

Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome in North America, has decimated bat populations within a decade. The fungus impacts bats during hibernation when physiological functions, including immune responses, are down-regulated. Studies have shown that Pd is native to Europe, where it is not associated with mass mortalities. Moreover, genomic and proteomic studies indicated that European bats may have evolved an effective immune defence, which is lacking in North American bats. However, it is still unclear which defence strategy enables European bats to cope with the pathogen. Here, we analyzed selected physiological and immunological parameters in torpid, Pd infected European greater mouse-eared bats (Myotis myotis) showing three different levels of infection (asymptomatic, mild and severe symptoms). From a subset of the studied bats we tracked skin temperatures during one month of hibernation. Contrasting North American bats, arousal patterns remained unaffected by Pd infections in M. myotis. In general, heavier M. myotis aroused more often from hibernation and showed less severe disease symptoms than lean individuals; most likely because heavy bats were capable of reducing the Pd load more effectively than lean individuals. In the blood of severely infected bats, we found higher gene expression levels of an inflammatory cytokine (IL-1β), but lower levels of an acute phase protein (haptoglobin), reactive oxygen metabolites (ROMs) and plasma non-enzymatic antioxidant capacity (OXY) compared to conspecifics with lower levels of infection. We conclude that M. myotis, and possibly also other European bat species, tolerate Pd infections during torpor by using selected acute phase response parameters at baseline levels, yet without arousing from torpor and without synthesizing additional immune molecules.

Plasma Non-Enzymatic Antioxidant Capacity (NEAC) in Relation to Dietary NEAC, Nutrient Antioxidants and Inflammation-Related Biomarkers.

(1) Background: Little is known about the interlinkages between dietary and plasma non-enzymatic antioxidant capacity (D-NEAC and P-NEAC, respectively) and the body’s antioxidant and inflammation response. Our aim was to explore these associations in 210 participants from two Spanish European Prospective Investigation into Cancer and Nutrition (EPIC) centers. (2) Methods: D-NEAC was estimated using published NEAC values in food. P-NEAC and total polyphenols (TP) were quantified by FRAP (ferric-reducing antioxidant power), TRAP (total radical-trapping antioxidant parameter), TEAC-ABTS (trolox equivalent antioxidant capacity-Azino Bis Thiazoline Sulfonic), ORAC (oxygen radical absorbance capacity) and Folin–Ciocalteu assays. Nutrient antioxidants (carotenes, α-tocopherol, ascorbic acid, retinol, uric acid, Q9 and Q10 coenzymes) and inflammation markers (IL-6, IL-8, CRP, TNF-α, PAI-I, resistin and adiponectin) were also analyzed. Spearman correlation and linear regression analyses were performed in association analyses. Analyses were stratified by covariates and groups were defined using cluster analysis. (3) Results: P-FRAP was correlated with D-NEAC, and significantly associated with P-NEAC in multivariate adjusted models. P-FRAP levels were also significantly associated with plasma antioxidants (log2 scale: TP β = 0.26; ascorbic acid β = 0.03; retinol β = 0.08; α-tocopherol β = 0.05; carotenes β = 0.02; Q10 β = 0.06; uric acid β = 0.25), though not with inflammation-related biomarkers. Different profiles of individuals with varying levels of P-NEAC and biomarkers were found. (4) Conclusions: P-NEAC levels were to some extent associated with D-NEAC and plasma antioxidants, yet not associated with inflammation response.

Carbonic anhydrase inhibition and chemoreflex control of breathing: A litmus test for methazolamide as a viable alternative to acetazolamide.

Carbonic anhydrase inhibition and chemoreflex control of breathing: A litmus test for methazolamide as a viable alternative to acetazolamide.

Impacts of land use on an insectivorous tropical bat: The importance of mercury, physio-immunology and trophic position

Deforestation, agricultural intensification, and habitat homogenization are critical threats to biodiversity in Southeast Asia. Limited information is available on the trophic and physiological responses of tropical animals to these environmental changes. The wrinkle-lipped free-tailed bat Chaerephon plicatus is a cave roosting species that is experiencing population declines across Southeast Asia, where landscapes have been drastically modified. In our study site in central Thailand, we tested the hypothesis that wrinkle-lipped free-tailed bats living in landscapes that contrast in heterogeneity and land-use differed in mercury contamination, trophic position and physio-immunological status. Bats from less heterogeneous landscapes (dominated by rice crops, absence of large forest patches) occupied a lower trophic position than conspecifics from more heterogeneous landscapes (including large forest patches). Additionally, bats from these habitats had lower concentrations of mercury in erythrocytes, lower body mass, higher antioxidant superoxide dismutase (SOD), lower antioxidant glutathione peroxidase (GPx) and lower values of the GPx/SOD ratio than bats from more heterogeneous landscapes. Individual bat mercury concentrations were positively correlated with body mass and two immune markers (lysozyme and immunoglobulin) but were negatively correlated with plasma non-enzymatic antioxidant capacity. Our results suggest various links among landscape heterogeneity, mercury exposure/accumulation, and health status of wildlife in Southeast Asian countries.

Ferric reducing ability of plasma and assessment of selected plasma antioxidants in adults with celiac disease.

Oxidative stress with an excessive free radical production and a reduction in the activity of protective antioxidants is considered as one of the mechanisms responsible for gluten toxicity. However, its role in celiac disease (CD) is unclear. Evaluation of plasma nonenzymatic antioxidant capacity in patients with CD (both untreated patients and those receiving gluten-free diet [GFD]) by measuring the ferric reducing ability of plasma (FRAP) as well as assessing selected plasma antioxidants. The study included 169 adult patients: 48 patients with untreated active CD, 72 patients with CD on a GFD, and 49 healthy controls. In each group, we measured the serum levels of selected antioxidants (uric acid, bilirubin, albumin, and vitamin E) and used the FRAP assay to assess the total antioxidant capacity (TAC) of plasma. In each patient, serological and histopathological activity of CD was also evaluated. There were no significant differences in the TAC of plasma measured with the FRAP assay between the study groups. Patients with CD had higher uric acid levels compared with controls (p <0.001), while bilirubin levels were lower in patients with active disease than in controls (p <0.05). Serum vitamin E levels were lower in all patients with CD compared with controls (p <0.01). The FRAP assay is not the method of choice for assessing the TAC of plasma in patients with CD. Owing to high serum uric acid levels, the FRAP assay results in these patients may be overestimated despite the reduced levels of other plasma antioxidants.

Non-enzymatic antioxidant capacity (NEAC) estimated by two different dietary assessment methods and its relationship with NEAC plasma levels.

We aimed to quantify and compare dietary non-enzymatic antioxidant capacity (NEAC), estimated using two dietary assessment methods, and to explore its relationship with plasma NEAC. Fifty healthy subjects volunteer to participate in this study. Two dietary assessment methods [a food frequency questionnaire (FFQ) and a 24-hour recall (24-HR)] were used to collect dietary information. Dietary NEAC, including oxygen radical absorbance capacity (ORAC), total polyphenols, ferric-reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity, was estimated using several data sources of NEAC content in food. NEAC status was measured in fasting blood samples using the same assays. We performed nonparametric Spearman's correlation analysis between pairs of dietary NEAC (FFQ and 24-HR) and diet-plasma NEAC, with and without the contribution of coffee's NEAC. Partial correlation analysis was used to estimate correlations regardless of variables potentially influencing these relationships. FFQ-based NEAC and 24-HR-based NEAC were moderately correlated, with correlation coefficients ranging from 0.54 to 0.71, after controlling for energy intake, age and sex. Statistically significant positive correlations were found for dietary FRAP, either derived from the FFQ or the 24-HR, with plasma FRAP (r~0.30). This weak, albeit statistically significant, correlation for FRAP was mostly present in the fruits and vegetables food groups. Plasma ORAC without proteins and 24-HR-based total ORAC were also positively correlated (r=0.35). The relationship between dietary NEAC and plasma FRAP and ORAC suggests the dietary NEAC may reflect antioxidant status despite its weak in vivo potential, supporting further its use in oxidative stress-related disease epidemiology.

The Peroxidation of Leukocytes Index Ratio Reveals the Prooxidant Effect of Green Tea Extract.

Despite tea increased plasma nonenzymatic antioxidant capacity, the European Food Safety Administration (EFSA) denied claims related to tea and its protection from oxidative damage. Furthermore, the Supplement Information Expert Committee (DSI EC) expressed some doubts on the safety of green tea extract (GTE). We performed a pilot study in order to evaluate the effect of a single dose of two capsules of a GTE supplement (200 mg × 2) on the peroxidation of leukocytes index ratio (PLIR) in relation to uric acid (UA) and ferric reducing antioxidant potential (FRAP), as well as the sample size to reach statistical significance. GTE induced a prooxidant effect on leukocytes, whereas FRAP did not change, in agreement with the EFSA and the DSI EC conclusions. Besides, our results confirm the primary role of UA in the antioxidant defences. The ratio based calculation of the PLIR reduced the sample size to reach statistical significance, compared to the resistance to an exogenous oxidative stress and to the functional capacity of oxidative burst. Therefore, PLIR could be a sensitive marker of redox status.

Green Tea and Bone Marrow Transplantation: From Antioxidant Activity to Enzymatic and Multidrug-resistance Modulation

Epigallocatechin-3-gallate (EGCG), the main flavonoid of green tea (GT), could play an active role in the prevention of oxidative-stress-related diseases, such as hematologic malignancies. Some effects of EGCG are not imputable to antioxidant activity, but involve modulation of antioxidant enzymes and uric acid (UA) levels. The latter is the major factor responsible of the plasma non-enzymatic antioxidant capacity (NEAC). However, hyperuricemia is a frequent clinical feature caused by tumor lysis syndrome or cyclosporine side effects, both before and after bone marrow transplantation (BMT). Besides this, food–drug interactions could be associated with GT consumption and could have clinical implications.The molecular mechanisms involved in the redox and drug metabolizing/transporting pathways were discussed, with particular reference to the potential role of GT and EGCG in BMT. Moreover, on reviewing data on NEAC, isoprostanes, uric acid, and various enzymes from human studies on GT, its extract, or EGCG, an increase in NEAC, without effect on isoprostanes, and contrasting results on UA and enzymes were observed.Currently, few and contrasting available evidences suggest caution for GT consumption in BMT patients and more studies are needed to better understand the potential impact of EGCG on oxidative stress and metabolizing/transporting systems.

Effect of the co-occurring components from olive oil and thyme extracts on the antioxidant status and its bioavailability in an acute ingestion in rats

The aim of this work was to examine whether bioactives in thyme could enhance the antioxidant capacity of phenolics in virgin olive oil and their bioavailability in Wistar rats. After acute oral administration of extracts from olive cake (OE), thyme (TE) or their combination (OTE), blood samples were collected from 0 to 360 min. Plasma antioxidant status was analyzed by DPPH and FRAP in plasma and by SOD, CAT and GPx activities in erythrocytes. Plasma pharmacokinetics of the main metabolites of bioactives in olive oil and thyme were characterized. Plasma non-enzymatic antioxidant capacity was significantly modulated by OE, TE, and OTE in a time-, assay, and extract-dependent manner. OE, TE, and OTE all significantly decreased superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity and catalase (CAT) activity was increased. Pharmacokinetic results showed that plasma concentration (Cmax) of the main olive phenolic metabolites in rats fed with OTE were similar to those of OE. These results indicate that an enhanced bioavailability of olive phenolic compounds could occur in the presence of thyme, although any synergistic effect was observed in the antioxidant status when both phenolic extracts were administered. Antioxidant protection by phenolics from olive and thyme against oxidative stress occurs primarily through a direct antioxidant effect and may be related to the phenolic plasmatic metabolites.

Effect of plant foods and beverages on plasma non-enzymatic antioxidant capacity in human subjects: a meta-analysis

Non-enzymatic antioxidant capacity (NEAC) represents a sensitive biomarker measuring the in vivo antioxidant potential of vegetable foods. To evaluate the effectiveness of plant-derived foods and beverages on the plasma non-enzymatic antioxidant system, we analysed all literature published upto May 2010. Data were extracted by two authors independently, and the effect size was summarised using standardised mean differences by a random-effects model. For the analysis, eighty-eight studies were included, reporting a total number of 122 interventions and involving 2890 subjects. There was overall evidence of the effectiveness of fruit, vegetables, dietary patterns based on plant foods, red wine and tea in increasing plasma NEAC. No changes were found for chocolate and fruit juices. We observed an overall effect size three times higher in subjects with risk factors when compared with healthy subjects. Total radical-trapping antioxidant parameter, oxygen radical absorbance capacity and ferric-reducing antioxidant power methods showed a similar increase in plasma NEAC following dietary supplementation, whereas Trolox equivalent antioxidant capacity did not respond to dietary supplementation. Data from the present meta-analysis show that plant-derived foods represent an effective strategy to enhance an endogenous antioxidant network in humans. This is particularly evident in the presence of oxidative stress-related risk factors.

Mediterranean diet and non enzymatic antioxidant capacity in the PREDIMED study: Evidence for a mechanism of antioxidant tuning

Background and aimsThe intake of antioxidant-rich foods may increase the blood levels of non enzymatic antioxidant capacity (NEAC). NEAC takes into account all antioxidants from food and synergistic effects between them. We examined the effect of a 1-year intervention with Mediterranean diet on plasma NEAC and assessed whether it was related to baseline NEAC levels. Methods and resultsFive hundred sixty-four participants at high cardiovascular risk were randomly selected from the PREDIMED (Prevención con DIeta MEDiterránea) Study, a large 3-arm randomized clinical trial. Blood NEAC levels were measured at baseline and after 1-year of dietary intervention with 1) a Mediterranean diet supplemented with virgin olive oil (MED + VOO); 2) a Mediterranean diet supplemented with nuts (MED + nuts), or 3) a control low-fat diet. Plasma NEAC was analyzed using FRAP (ferric reducing antioxidant potential) and TRAP (total radical-trapping antioxidant parameter) assays. Plasma FRAP levels increased after 1-year of intervention with MED + VOO [72.0 μmol/L (95% CI, 34.2–109.9)] and MED + nuts [48.9 μmol/L (24.3–73.5)], but not after the control low-fat diet [13.9 μmol/L (−11.9 to 39.8)]. Participants in the lowest quartile of plasma FRAP at baseline significantly increased their levels after any intervention, while those in the highest quartile decreased. Similar results occurred with TRAP levels. ConclusionsThis study shows that a 1-year of MED diet intervention increases plasma TAC level in subjects at high risk for cardiovascular disease. Moreover, the effectiveness of dietary supplementation with antioxidants may be related to baseline levels of plasma NEAC.

Quercetin attenuates oxidative stress in the blood plasma of rats bearing DMBA-induced mammary cancer and treated with a combination of doxorubicin and docetaxel

The development of side-effects during doxorubicin-docetaxel (DOX-DTX) chemotherapy is considered as related to generation of oxidative stress by DOX. The addition of docetaxel potentiates this effect. Thus, antioxidants are assumed as a promising remedy for neutralizing deteriorating effects of reactive oxygen species (ROS) in pathological conditions and polyphenolic antioxidants are suitable candidates for such a therapeutic approach. We evaluated the ability of quercetin to attenuate oxidative stress developed during the process of DMBA carcinogenesis and DOX-DTX chemotherapy in the blood plasma of rats bearing mammary tumors. We have found that quercetin significantly improved the plasma nonenzymatic antioxidant capacity (NEAC) and reduced lipid peroxidation, which suggest the beneficial effect of flavonoid. The inclusion of quercetin to the DOX-DTX chemotherapy was also advantageous. A considerable decrease of carbonyls and lipid peroxidation products (TBARS) and improvement of the endogenous antioxidant defense system (an increase of NEAC, thiols and SOD activity) were observed compared to rats treated with DOX-DTX chemotherapy. These results suggest that quercetin could protect blood plasma constituents against oxidative damage evoked by DOX and DTX.

Interaction between sexual steroids and immune response in affecting oxidative status of birds

One hypothesis explaining the honesty of secondary sexual traits regulated by testosterone (T) is that T can impair the balance between pro-oxidant compounds and antioxidant defences, favouring a status of oxidative stress that only good quality individuals can sustain (oxidative handicap hypothesis). In the present study, we evaluated for the first time the effects of sexual steroids, T and its metabolites 5-α-dihydrotestosterone (DHT) and estradiol (E2) on oxidative damage and plasma non-enzymatic antioxidant capacity, while birds are faced by an oxidative challenge induced by an immune stimulation with sheep red blood cells. We used male and female diamond doves Geopelia cuneata, a species that shows an orange-red periorbital ring, whose size and color are strongly affected by androgens, but not by estrogens. Immunization increased oxidative damage in all groups, regardless of hormone treatment. It also decreased anti-oxidant capacity in all groups, except for testosterone treated birds. The ratio of oxidative damage over anti-oxidant capacity (oxidative stress) was increased in both immunological challenged controls and E2 birds, while challenged birds treated with androgens did not differ from non-challenged birds. The response of males and females to our treatments never differed. Our results undermine the idea that T can induce honest signalling through a pro-oxidant activity.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Interplay between plasma oxidative status, cortisol and coping styles in wild alpine marmots, Marmota marmota

Variation in how individuals cope behaviourally and physiologically with stressors is widespread and can have a significant impact on life-history traits and fitness. Individual coping styles are characterised by differential behavioural and adrenocortical reactivity to various challenges. As stress hormones can affect the production of reactive chemical species and the antioxidant status, individuals with different coping styles may differ also in oxidative status. Field studies on wild mammalian populations are few in number and none so far has simultaneously tested the relationship between coping style, adrenocortical reactivity and oxidative status in the same individuals. We measured individual variation in coping styles along a proactive-reactive continuum together with variation in baseline and stress-induced plasma oxidative damage, plasma non-enzymatic antioxidant capacity and cortisol in wild alpine marmots, Marmota marmota. Confirmatory path analysis revealed that different coping styles are accompanied by different baseline and stress-induced plasma oxidative statuses. Our findings also highlight the potential role of cortisol as a mediator of such differences.

Variation of a carotenoid-based trait in relation to oxidative stress and endocrine status during the breeding season in the Eurasian kestrel: A multi-factorial study

Carotenoid-based skin colorations vary seasonally in many bird species and are thought to be honest sexually selected signals. In order to provide more insight in the potential signal function and underlying mechanisms of such colorations we here quantified patterns of variation of leg coloration in adult male and female Eurasian kestrels ( Falco tinnunculus tinnunculus) over the breeding season, and evaluated the relationship between coloration and levels of carotenoids, androgens and estrogens, oxidative damage and plasma non-enzymatic antioxidant capacity. We studied both reproducing wild and non-reproducing captive birds to test for the effect of diet and breeding effort. Males were more colored than females only during mating, and independently of diet, suggesting that leg-color is a sexually selected trait. Seasonal variation in leg color was associated with circulating carotenoids, but concentrations of these molecules were not related to antioxidant capacity, body condition or oxidative damage. These results indicate that carotenoid-based colorations may not be an honest signal of health status in this species. Production of carotenoid rich eggs coincided with low levels of circulating carotenoids in females, indicating that carotenoids might be a limited resource for laying female kestrels. Finally, young rearing males had higher levels of oxidative damage than females, and wild birds of both sexes had higher levels of these parameters than captive birds. These results may indicate that parental effort and physical activity are costly, independently from hormonal status. Since androgens did not explain carotenoid variation we suggest that multiple interacting factors can regulate carotenoid levels along the season.