Comparative Metabolomics of Ligulate and Tubular Flowers of Two Cultivars of Calendula officinalis L.

Calendula officinalis L. is a well-known plant widely used in traditional medicine due to the presence of various biologically active compounds. The main raw material for the production of medicinal preparations is the inflorescence, which consists of ligulate and tubular flowers. However, the characteristics of the metabolome of these flowers are not fully understood. This study identified and compared the levels of major metabolites in the ligulate and tubular flowers of two C. officinalis cultivars, 'Golden Sea' (GS) and 'Paradise Garden' (PG). The metabolome was analysed using ultra-performance liquid chromatography with photodiode array detection and a Q Exactive Orbitrap high-resolution mass spectrometer. It was found that the tubular flowers of both PG and GS cultivars had higher levels of lipids, phenolamides and caffeoylquinic acids and lower levels of triterpenoid glycosides than the ligulate flowers. It was also shown that the inflorescences of the GS, which had a 35% higher proportion of tubular flowers, contained 30% more phenolic compounds and 50% more lipids than the PG. Thus, the results obtained extend our understanding of the features in the metabolomes of ligulate and tubular flowers and suggest that the quality of inflorescences of C. officinalis cultivars, as a source of medicinal preparations, is strongly influenced by the proportion of ligulate and tubular flowers.

Mass Spectrometric Fingerprint Mapping Reveals Species-Specific Differences in Plant Polyphenols and Related Bioactivities.

Plant species show large variation in the composition and content of their tannins and other polyphenols. These large metabolites are not easy to measure accurately, but they are important factors for species bioactivity and chemotaxonomy. Here, we used an automated group-specific UHPLC-DAD-MS/MS tool to detect and quantify eight most common polyphenol groups in 31 chemically diverse plant species representing many types of growth forms and evolutionary ages. Ten replicate plants were used for each species and two polyphenol-related bioactivities, i.e., protein precipitation capacity and oxidative activity were measured in all samples as well. By the help of a novel 2D fingerprint mapping tool we were able to visualize the qualitative and quantitative differences between the species in hydrolysable tannins (galloyl and hexahydroxydiphenoyl derivatives), proanthocyanidins (procyanidins and prodelphinidins), flavonols (kaempferol, quercetin and myricetin derivatives) and quinic acid derivatives together with the two bioactivities. The highest oxidative activities were found with species containing ellagitannins (e.g., Quercus robur, Geranium sylvaticum, Lythrum salicaria and Chamaenerion angustifolium) or prodelphinidin-rich proanthocyanidins (e.g., Ribes alpinum, Salix phylicifolia and Lysimachia vulgaris). The best species with high protein precipitation capacity were rich in gallotannins (Acer platanoides and Paeonia lactiflora) or oligomeric ellagitannins (e.g., Comarum palustre, Lythrum salicaria and Chamaenerion angustifolium). These types of tools could prove their use in many types of screening experiments and might reveal even unusually active polyphenol types directly from the crude plant extracts.

Alkaline oxidization can increase the in vitro antiparasitic activity of proanthocyanidin-rich plant extracts against Ascarissuum.

Proanthocyanidins (PAs) are a class of plant specialized metabolites with well-documented bioactivities such as antiparasitic effects. However, little is known about how the modification of PAs influences their bioactivity. The objective of this study was to investigate a wide range of PA-containing plant samples to determine if extracts containing PAs modified by oxidation had altered antiparasitic activities, compared to the original extracts that had not been modified in alkaline conditions. We extracted and analyzed samples from 61 proanthocyanidin-rich plants. The extracts were then oxidized under alkaline conditions. We used these non-oxidized and oxidized proanthocyanidin-rich extracts to conduct a detailed analysis of direct antiparasitic effects against the intestinal parasite Ascaris suum in vitro. These tests showed that the proanthocyanidin-rich extracts had antiparasitic activity. Modification of these extracts significantly increased the antiparasitic activity for the majority the extracts, suggesting that the oxidation procedure enhanced the bioactivity of the samples. Some samples that showed no antiparasitic activity before oxidation showed very high activity after the oxidation. High levels of other polyphenols in the extracts, such as flavonoids, was found to be associated with increased antiparasitic activity following oxidation. Thus, our in vitro screening opens up the opportunity for future research to better understand the mechanism of action how alkaline treatment of PA-rich plant extracts increases their biological activity and potential as novel anthelmintics.

Comparative analysis of the anthelmintic efficacy of European heather extracts on Teladorsagia circumcincta and Trichostrongylus colubriformis egg hatching and larval motility.

BACKGROUND: Gastrointestinal nematode (GIN) control is traditionally achieved with the use of anthelmintic drugs, however due to regulations in organic farming and the rise in anthelmintic resistance, alternatives are sought after. A promising alternative is the use of bioactive plant feeding due to the presence of plant secondary metabolites (PSMs) such as proanthocyanidins (PAs). This study focussed on the perennial shrub heather (Ericaceae family), a plant rich in PAs, highly abundant across Europe and with previously demonstrated anthelmintic potential. METHODS: In vitro assays were used to investigate heather's anthelmintic efficacy against egg hatching and larval motility. Heather samples were collected from five European countries across two seasons, and extracts were tested against two GIN species: Teladorsagia circumcincta and Trichostrongylus colubriformis. Polyphenol group-specific ultraperformance liquid chromatography-tandem mass spectrometry analysis was performed to identify relevant polyphenol subgroups present, including the PA concentration and size and ratio of the subunits. Partial least squares analysis was performed to associate efficacy with variation in PSM composition. RESULTS: Heather extracts reduced egg hatching of both GIN species in a dose-dependent manner by up to 100%, while three extracts at the highest concentration (10 mg/ml) reduced larval motility to levels that were not significantly different from dead larvae controls. PAs, particularly the procyanidin type, and flavonol derivatives were associated with anthelmintic activity, and the particular subgroup of polyphenols associated with the efficacy was dependent on the GIN species and life stage. CONCLUSIONS: Our results provide in vitro evidence that heather, a widely available plant often managed as a weed in grazing systems, has anthelmintic properties attributed to various groups of PSMs and could contribute to sustainable GIN control in ruminant production systems across Europe.

Chemotaxonomic Markers for the Leaf Buds of Common Finnish Trees and Shrubs: A Rapid UHPLC MS Fingerprinting Tool for Species Identification.

In this study, a chemotaxonomic tool was created on the basis of ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) for the identification of 13 common Finnish deciduous trees and shrubs from their leaf bud metabolites. The bud extracts were screened with UHPLC-ESI-QqQ-MS and UHPLC-ESI-Q-Orbitrap-MS to discover suitable markers for each species. Two approaches were tested in the marker selection: (1) unique species-specific markers to obtain selective fingerprints per species and (2) major markers to maximise the sensitivity of the fingerprints. The markers were used to create two selected ion-recording-based fingerprinting tools with UHPLC-ESI-QqQ-MS. The methods were evaluated for their selectivity, repeatability, and robustness in plant species identification by analysing leaf buds from several replicates of each species. The created chemotaxonomic tools were shown to provide unique chromatographic profiles for the studied species in less than 6 min. A variety of plant metabolites, such as flavonoids, triterpenoids, and hydroxycinnamic acid derivatives, were found to serve as good chemotaxonomic markers for the studied species. In 10 out of 13 cases, species-specific markers were superior in creating selective and repeatable fingerprints.

Dietary proanthocyanidins promote localized antioxidant responses in porcine pulmonary and gastrointestinal tissues during Ascaris suum-induced type 2 inflammation.

Proanthocyanidins (PAC) are dietary polyphenols with putative anti-inflammatory and immunomodulatory effects. However, whether dietary PAC can regulate type-2 immune function and inflammation at mucosal surfaces remains unclear. Here, we investigated if diets supplemented with purified PAC modulated pulmonary and intestinal mucosal immune responses during infection with the helminth parasite Ascaris suum in pigs. A. suum infection induced a type-2 biased immune response in lung and intestinal tissues, characterized by pulmonary granulocytosis, increased Th2/Th1 T cell ratios in tracheal-bronchial lymph nodes, intestinal eosinophilia, and modulation of genes involved in mucosal barrier function and immunity. Whilst PAC had only minor effects on pulmonary immune responses, RNA-sequencing of intestinal tissues revealed that dietary PAC significantly enhanced transcriptional responses related to immune function and antioxidant responses in the gut of both naïve and A. suum-infected animals. A. suum infection and dietary PAC induced distinct changes in gut microbiota composition, primarily in the jejunum and colon, respectively. Notably, PAC consumption substantially increased the abundance of Limosilactobacillus reuteri. In vitro experiments with porcine macrophages and intestinal epithelial cells supported a role for both PAC polymers and PAC-derived microbial metabolites in regulating oxidative stress responses in host tissues. Thus, dietary PAC may have distinct beneficial effects on intestinal health during infection with mucosal pathogens, while having a limited activity to modulate naturally-induced type-2 pulmonary inflammation. Our results shed further light on the mechanisms underlying the health-promoting properties of PAC-rich foods, and may aid in the design of novel dietary supplements to regulate mucosal inflammatory responses in the gastrointestinal tract.

Large Inter- and Intraspecies Variability of Polyphenols and Proanthocyanidins in Eight Temperate Forage Species Indicates Potential for Their Exploitation as Nutraceuticals.

Substantial efforts have been made in incorporating tannin-rich forages into grassland-based livestock production systems. However, the structural and functional diversity of tannins in different species limits their potential use at the field scale. We conducted a greenhouse experiment with 17 cultivars from 8 forage species and their cultivars. Ultraperformance liquid chromatography tandem mass spectrometry was used to analyze their polyphenolic profile and proanthocyanidin (PA) structural features in leaves. Our results highlight large inter- and intraspecies variability of plants in terms of polyphenol and tannin concentrations in the leaves. A concomitant and significant variation was also registered in the structural features of PA-rich forages such as the mean degree of polymerization and prodelphinidin percentage. The concentration of PA also varied within plant organs; the highest concentration was in flowers, but leaves had the highest contribution to harvestable PA biomass. Our research highlights that identifying these variations helps in identifying the representativeness of bioactivity and provides the basis for targeted breeding programs.

Structure-function analysis of purified proanthocyanidins reveals a role for polymer size in suppressing inflammatory responses.

Proanthocyanidins (PAC) are dietary compounds that have been extensively studied for beneficial health effects due to their anti-inflammatory properties. However, the structure-function relationships of PAC and their mode-of-action remain obscure. Here, we isolated a wide range of diverse PAC polymer mixtures of high purity from plant material. Polymer size was a key factor in determining the ability of PAC to regulate inflammatory cytokine responses in murine macrophages. PAC polymers with a medium (9.1) mean degree of polymerization (mDP) induced substantial transcriptomic changes, whereas PAC with either low (2.6) or high (12.3) mDP were significantly less active. Short-term oral treatment of mice with PAC modulated gene pathways connected to nutrient metabolism and inflammation in ileal tissue in a polymerization-dependent manner. Mechanistically, the bioactive PAC polymers modulated autophagic flux and inhibited lipopolysaccharide-induced autophagy in macrophages. Collectively, our results highlight the importance of defined structural features in the health-promoting effects of PAC-rich foods.

Characterization of Natural and Alkaline-Oxidized Proanthocyanidins in Plant Extracts by Ultrahigh-Resolution UHPLC-MS/MS.

In this study, we analyzed the proanthocyanidin (PA) composition of 55 plant extracts before and after alkaline oxidation by ultrahigh-resolution UHPLC-MS/MS. We characterized the natural PA structures in detail and studied the sophisticated changes in the modified PA structures and the typical patterns and models of reactions within different PA classes due to the oxidation. The natural PAs were A- and B-type PCs, PDs and PC/PD mixtures. In addition, we detected galloylated PAs. B-type PCs in different plant extracts were rather stable and showed no or minor modification due to the alkaline oxidation. For some samples, we detected the intramolecular reactions of PCs producing A-type ether linkages. A-type PCs were also rather stable with no or minor modification, but in some plants, the formation of additional ether linkages was detected. PAs containing PD units were more reactive. After alkaline oxidation, these PAs or their oxidation products were no longer detected by MS even though a different type and/or delayed PA hump was still detected by UV at 280 nm. Galloylated PAs were rather stable under alkaline oxidation if they were PC-based, but we detected the intramolecular conversion from B-type to A-type. Galloylated PDs were more reactive and reacted similarly to nongalloylated PDs.

Modification of Natural Proanthocyanidin Oligomers and Polymers Via Chemical Oxidation under Alkaline Conditions.

We tested the susceptibility of 102 proanthocyanidin (PA)-rich plant extracts to oxidation under alkaline conditions and the possibility to produce chemically modified PAs via oxidation. Both the nonoxidized and the oxidized extracts were analyzed using group-specific ultrahigh-performance liquid chromatography-diode array detection-tandem mass spectrometry (UHPLC-DAD-MS/MS) methods capable of detecting procyanidin (PC) and prodelphinidin (PD) moieties along the two-dimensional (2D) chromatographic fingerprints of plant PAs. The results indicated different reactivities for PCs and PDs. When detected by UHPLC-DAD only, most of the PC-rich samples exhibited only a subtle change in their PA content, but the UHPLC-MS/MS quantitation showed that the decrease in the PC content varied by 0-100%. The main reaction route was concluded to be intramolecular. The PD-rich and galloylated PAs showed a different pattern with high reductions in the original PA content by both ultraviolet (UV) and MS/MS quantitation, accompanied by the shifted retention times of the chromatographic PA humps. In these samples, both intra- and intermolecular reactions were indicated.

The Effect of Growth Medium Strength on Minimum Inhibitory Concentrations of Tannins and Tannin Extracts against E. coli.

In this study the effect of growth medium strength on the minimum inhibitory concentration (MIC) of different tannins and tannin extracts against Escherichia coli was systematically investigated for the first time. Three pure compounds (vescalagin, castalagin and gallic acid) and five extracts (chestnut, quebracho, mimosa, Colistizer and tannic acid) were studied. Broth microdilution was assayed and bacteria were grown using different growth medium strengths varying from half to double the concentration recommended by the producer. MICs were determined using the iodonitrotetrazolium chloride (INT) dye or turbidity measurements. It was observed that MIC values depend on the growth medium strength. With an increase in the growth medium concentration MIC values rose roughly linearly for all samples, while their relative order remained unchanged, indicating that a direct interaction of tannins with growth medium nutrients represents the likely source of their antimicrobial activity. Understanding the effect of growth medium strength can finally yield a plausible explanation for the observed variation in MIC values reported in the scientific literature as well as provide help in planning proper applications of tannins in the livestock production.

Low Concentrations of Protein- and Fiber-Bound Proanthocyanidins in Sainfoin (Onobrychis viciifolia) Are Stable across Accessions, Growth Stages, and Drought Conditions.

Proanthocyanidins (PAs) in forages may be present in either soluble (S-PA) or non-extractable (NE-PA) form. Despite potential benefits of the NE-PA in ruminant nutrition, few studies have analyzed NE-PA in forages. This study examined the impact of a range of conditions on S-PA as well as protein- and fiber-bound PA (the NE-PA fractions) in sainfoin (Onobrychis viciifolia). Thus, five sainfoin accessions in either generative or vegetative stage were subjected to drought for 18 weeks and sampled repeatedly for PA analysis. Drought-stressed plants increased S-PAs on average by 59% across all accessions yet only in the vegetative stage. In contrast, NE-PA concentrations were generally lower (on average 15% of the total PAs) and unaffected by drought. Thus, for sainfoin, the low and stable concentration of NE-PAs across accessions, growth stages, and drought conditions should have a low, predictable impact on the future sainfoin analyses and feeding studies.

Glyphosate-based herbicide has soil-mediated effects on potato glycoalkaloids and oxidative status of a potato pest.

Glyphosate is the most used herbicide worldwide, targeting physiological pathways in plants. Recent studies have shown that glyphosate can also cause toxic effects in animals. We investigated the glyphosate-based herbicide (GBH)-induced changes in potato (Solanum tuberosum) plant chemistry and the effects of a GBH on the survival rate and oxidative status of the Colorado potato beetle (Leptinotarsa decemlineata). The beetles were reared on potato plants grown in pots containing soil treated with a GBH (Roundup Gold, 450 g/l) or untreated soil (water control). The 2nd instar larvae were introduced to the potato plants and then collected in 2 phases: as 4th instar larvae and as adults. The main glycoalkaloids of the potato plants, α-solanine and α-chaconine, were measured twice during the experiment. The α-solanine was reduced in potato plants grown in GBH-treated soil, which can be detrimental to plant defenses against herbivores. GBH treatment had no effect on the survival rate or body mass of the larvae or the adult beetles. In the larvae, total glutathione (tGSH) concentration and the enzyme activity of catalase (CAT), superoxide dismutase, and glutathione-S-transferase were increased in the GBH treatment group. In the adult beetles, CAT activity and tGSH levels were affected by the interactive effect of GBH treatment and the body mass. To conclude, environmentally relevant concentrations of a GBH can affect the potato plant's glycoalkaloid concentrations, but are not likely to directly affect the survival rate of the Colorado potato beetle, but instead, modify the antioxidant defense of the beetles via diet.

Ellagitannins Inhibit the Exsheathment of Haemonchus contortus and Trichostrongylus colubriformis Larvae: The Efficiency Increases Together with the Molecular Size.

Worldwide, parasitic gastrointestinal nematodes continue to threaten animal health, welfare, and production in outdoor breeding systems of small ruminants. For more than 50 years, the control of these parasitic worms has relied on the use of commercial synthetic anthelmintics. However, anthelmintic resistance in worm populations is nowadays widespread and requires novel solutions. The use of tannin-rich plants has been suggested as an alternative to synthetic anthelmintics to control gastrointestinal nematodes. The majority of previous studies have focused on the activity of proanthocyanidins (syn condensed tannins), and less is known about ellagitannins. In this study, the effects of 30 structurally unique ellagitannins on the exsheathment of third-stage infective larvae were examined on Haemonchus contortus and Trichostrongylus colubriformis by the in vitro larval exsheathment inhibition assay. Ellagitannins were found to be promising natural anthelmintics as they showed direct inhibition on larval exsheathment for both nematode species. In general, ellagitannins were more efficient at inhibiting the exsheathment of H. contortus larvae than those of T. colubriformis. The efficiency of inhibition increased as the degree of oligomerization or the molecular weight of the ellagitannin increased. Otherwise, we found no other structural features of ellagitannins that significantly affected the anthelmintic activity on the third-stage infective larvae. The effective concentrations were physiologically relevant and should be achievable in the gastrointestinal tract also in in vivo conditions.

Isolation of chemically well-defined semipreparative liquid chromatography fractions from complex mixtures of proanthocyanidin oligomers and polymers.

In this study, a semipreparative liquid chromatography method was developed for the isolation of chemically well-defined proanthocyanidin (PA) oligomer and polymer fractions. The aim was to achieve better separation than traditionally achieved for the PAs with other chromatographic methods. The method was tested with eleven PA rich Sephadex LH-20 fractions, which originated from eleven different plant species. The resulting semipreparative fractions were analyzed by both triple quadrupole and high-resolution mass spectrometry assisted by ultrahigh-performance liquid chromatography (UPLC) separation. The results showed remarkable differences in the procyanidin to prodelphinidin ratio, mean degree of polymerization, and specific oligomeric and polymeric content. However, some of these features indicated consistent patterns between species as the function of UPLC retention time. The developed method enables the production of tens of well-defined fractions of PA oligomers and polymers from the unresolved chromatographic PA hump. Accordingly, this allows researchers to explore the most bioactive parts of the complex PA humps of any plant species, which have not been possible earlier.

Two-Dimensional Tannin Fingerprints by Liquid Chromatography Tandem Mass Spectrometry Offer a New Dimension to Plant Tannin Analyses and Help To Visualize the Tannin Diversity in Plants.

Tannins are large-molecular-weight plant polyphenols that are produced in fruits, berries, leaves, flowers, seeds, stems, and roots of woody and non-woody plants. Hundreds and thousands of individual tannin structures are consequently found in many kinds of natural food and feed products. The huge structural variability in tannins is reflected as vast bioactivity differences between them but not in the accuracy of their typical analysis methods. Here, I show how the modern liquid chromatography mass spectrometry methods can be used to obtain new types of two-dimensional tannin fingerprints to better visualize both the tannin content and diversity in plants with just one 10 min analysis per sample.

Oxidatively Active Plant Phenolics Detected by UHPLC-DAD-MS after Enzymatic and Alkaline Oxidation.

We developed a combination of methods to estimate the alkaline oxidative conditions of the midgut of insect larvae and to reveal the alkaline and enzymatic oxidative activities for individual phenolic compounds present in the larval host plants. First, we monitored the in vitro isomerization of 5-O-caffeoylquinic acid (5-CQA) into 3-CQA, 4-CQA and 5-CQA at pH 9.0-11.0. Then we calculated the isomer ratios of 3-CQA, 4-CQA and 5-CQA from the frass of eight species of insect herbivores fed on foliage containing 5-CQA. The isomer ratios suggested that the midgut pH of these larvae ranged from 9.4 to around 10.1. Second, we developed an in situ enzymatic oxidation method that enabled oxidation of phenolics in a frozen plant sample at 30 °C by species- and tissue-specific enzymes. Then we measured the alkaline and enzymatic oxidative activities of the individual phenolics in 20 plant species by quantifying the proportion of the compound concentration lost due to the auto-oxidation of a plant extract at pH 10 and due to the enzymatic oxidation of the frozen plant sample at 30 °C. Our results showed that both of the oxidative activity types depended primarily on the type of phenolic compound, but the enzymatic oxidative activity depended also on the plant species and tissue type. This combination of methods offers an approach to characterize a wide array of phenolics that are susceptible to oxidation by the plant enzymes and/or by the alkaline conditions estimated to prevail in the insect midgut. We propose that these kinds of compound-specific results could guide future studies on specific plant-herbivore interactions to focus on the phenolics that are likely to be active rather than inactive plant phenolics.

Variability in Foliar Ellagitannins of Hippophaë rhamnoides L. and Identification of a New Ellagitannin, Hippophaenin C.

Berries of common sea-buckthorn (Hippophaë rhamnoides L.) are well-known and used for their bioactive components, and while there is a considerable amount of research on the leaves as well, their ellagitannins (ETs) have not been a prominent focus of research. We identified and quantified ten major hydrophilic polyphenols, all ETs, in H. rhamnoides leaves and compared their abundance between 58 plant individuals. Of these compounds, hippophaenin C was characterized as a new ellagitannin by various spectrometric methods. The total concentrations of ETs ranged from 42.5 mg g-1 dry weight (DW) to 109.1 mg g-1 DW between individual plants. Among the ETs, hippophaenin C, stachyurin, and casuarinin were on average the most abundant compounds. Sexes did not differ significantly, while cultivars showed variation in some ETs. These results suggest that H. rhamnoides leaves could be a potential and rich source of several ETs.

Hydrolyzable Tannins, Flavonol Glycosides, and Phenolic Acids Show Seasonal and Ontogenic Variation in Geranium sylvaticum.

The seasonal variation of polyphenols in the aboveground organs and roots of Geranium sylvaticum in four populations was studied using UPLC-DAD-ESI-QqQ-MS/MS. The content of the main compound, geraniin, was highest (16% of dry weight) in the basal leaves after the flowering period but stayed rather constant throughout the growing season. Compound-specific mass spectrometric methods revealed the different seasonal patterns in minor polyphenols. Maximum contents of galloylglucoses and flavonol glycosides were detected in the small leaves in May, whereas the contents of further modified ellagitannins, such as ascorgeraniin and chebulagic acid, increased during the growing season. In flower organs, the polyphenol contents differed significantly between ontogenic phases so that maximum amounts were typically found in the bud phase, except in pistils the amount of gallotannins increased significantly in the fruit phase. These results can be used in evaluating the role of polyphenols in plant-herbivore interactions or in planning the best collection times of G. sylvaticum for compound isolation purposes.

Vitamin profiles in two free-living passerine birds under a metal pollution gradient - A calcium supplementation experiment.

Vitamin and carotenoid deficiency may impair development in free-living vertebrates, because of the importance of these micronutrients to growth, antioxidant defense and calcium regulation. Micronutrient and calcium insufficiency can be intensified by metal pollution which can interfere with nutrient homeostasis or indirectly reduce food availability. Furthermore, absorption of dietary heavy metals is dependent on food calcium and vitamin levels. We investigated the effect of calcium on plasma vitamin and carotenoid profiles and how these affected growth and survival in two passerine birds with different calcium turnover living along a metal pollution gradient. Vitamins (A, D3 and E) and carotenoids were quantified from blood plasma of great tit (Parus major) and pied flycatcher (Ficedula hypoleuca) nestlings. Metal concentrations in soil and in feces from the same nestlings were used to assess the exposure to air pollution. Additionally, we examined the vitamin level variation between developmental stages (eggs and nestlings within the same brood). Our results showed that generally higher concentrations of vitamins and carotenoids circulate in blood of great tits than in pied flycatchers. In general, birds inhabiting the polluted zone presented lower concentrations of the studied micronutrients. Calcium supplementation and metal pollution decreased vitamin A concentration in pied flycatcher, but not in great tit, while vitamin A affected growth and survival in great tit and pied flycatcher respectively. Our results suggest that populations under exposure to metal pollution may experience increased vitamin A deficiency, and that the two passerine species, while obtaining similar micronutrients in food, respond differently to environmental disturbance of nutrients.