pH-dependent nitration of para-hydroxyphenylacetic acid in the stomach.

The major urinary metabolite of nitrotyrosine is 3-nitro-4-hydroxyphenylacetic acid (3-Nitro-HPA). However, recent animal studies have shown that the majority of urinary 3-Nitro-HPA is derived from nitration of endogenous para-hydroxyphenylacetic acid (HPA), a metabolite of tyrosine. One potential site for the formation of 3-Nitro-HPA is the stomach, where nitrous acid is formed by the reaction of nitrite in saliva with gastric acid. The aim of this study was to determine whether there is pH-dependent nitration of salivary para-hydroxyphenylacetic acid or tyrosine, and the effects of dietary nitrate. Healthy volunteers (n = 18) ingested either a low or high nitrate diet, with and without the administration of omeprazole, a proton pump inhibitor. Urinary 3-Nitro-HPA excretion increased from 197 +/- 52 to 319 +/- 88 microg/day on switching from a low to a high nitrate diet (P < 0.05), and decreased (166 +/- 53 mug/day, P < 0.05) when gastric pH was increased by omeprazole. To determine whether 3-Nitro-HPA can be formed by nitration of para-hydroxyphenylacetic acid in the stomach, 500 microg of deuterated para-hydroxyphenylacetic acid was ingested with a high nitrate meal. This led to the excretion of both deuterated HPA and 3-Nitro-HPA in the urine, confirming that para-hydroxyphenylacetic acid is absorbed, and nitrated. Since omeprazole decreases the formation of 3-Nitro-HPA, presumably by decreasing the nitration of endogenous para-hydroxyphenylacetic acid present in saliva, and the observation that ingested deuterated para-hydroxyphenylacetic acid is nitrated and excreted, we conclude that endogenous para-hydroxyphenylacetic acid is nitrated in the stomach, absorbed, and excreted as 3-Nitro-HPA.

Nitration of endogenous para-hydroxyphenylacetic acid and the metabolism of nitrotyrosine.

Reactive nitrogen species, such as peroxynitrite, can nitrate tyrosine in proteins to form nitrotyrosine. Nitrotyrosine is metabolized to 3-nitro-4-hydroxyphenylacetic acid (NHPA), which is excreted in the urine. This has led to the notion that measurement of urinary NHPA may provide a time-integrated index of nitrotyrosine formation in vivo. However, it is not known whether NHPA is derived exclusively from metabolism of nitrotyrosine, or whether it can be formed by nitration of circulating para -hydroxyphenylacetic acid (PHPA), a metabolite of tyrosine. In the present study, we have developed a gas chromatography MS assay for NHPA and PHPA to determine whether or not NHPA can be formed directly by nitration of PHPA. Following the injection of nitrotyrosine, 0.5+/-0.16% of injected dose was recovered unchanged as nitrotyrosine, and 4.3+/-0.2% as NHPA in the urine. To determine whether or not NHPA could be formed by the nitration of PHPA, deuterium-labelled PHPA ([(2)H(6)]PHPA) was injected, and the formation of deuterated NHPA ([(2)H(5)]NHPA) was measured. Of the infused [(2)H(6)]PHPA, 78+/-2% was recovered in the urine unchanged, and approx. 0.23% was recovered as [(2)H(5)]NHPA. Since the plasma concentration of PHPA is markedly higher than free nitrotyrosine (approx. 400-fold), the nitration of high-circulating endogenous PHPA to form NHPA becomes very significant and accounts for the majority of NHPA excreted in urine. This is the first study to demonstrate that NHPA can be formed by nitration of PHPA in vivo, and that this is the major route for its formation.

Flavonoid permeability across an in situ model of the blood-brain barrier.

Understanding mechanisms associated with flavonoid neuroprotection is complicated by the lack of information on their ability to enter the CNS. This study examined naringenin and quercetin permeability across the blood-brain barrier (BBB), using in vitro (ECV304/C6 coculture) and in situ (rat) models. We report measurable permeabilities (P(app)) for both flavonoids across the in vitro BBB model, consistent with their lipophilicity. Both flavonoids showed measurable in situ BBB permeability. The rates of uptake (K(in)) into the right cerebral hemisphere were 0.145 and 0.019 ml min(-1) g(-1) for naringenin and quercetin, respectively. Quercetin K(in) was comparable to that of colchicine (0.006 ml min(-1) g(-1)), a substrate for P-glycoprotein (P-gp). Preadministration of the P-gp inhibitor PSC833 or GF120918 (10 mg/kg body wt) significantly increased colchicine K(in), but only GF120918 (able to inhibit breast cancer resistance protein, BCRP) affected K(in) for quercetin. Naringenin K(in) was not affected. The influence of efflux transporters on flavonoid permeability at the BBB was further studied using MDCK-MDR1 and immortalized rat brain endothelial cells (RBE4). Colchicine, quercetin, and naringenin all showed measurable accumulation (distribution volume, V(d) (microl/mg protein)) in both cell types. The V(d) for colchicine increased significantly in both cell lines following coincubation with either PSC833 (25 microM) or GF120918 (25 microM). Both inhibitors also caused an increase in naringenin V(d); by contrast only GF120918 coincubation significantly increased quercetin V(d). In conclusion, the results demonstrate that flavonoids are able to traverse the BBB in vivo. However, the permeability of certain flavonoids in vivo is influenced by their lipophilicity and interactions with efflux transporters.

Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products.

The metabolism of chlorogenic acid, naringin, and rutin, representative members of three common families of dietary polyphenols, the hydroxycinnamates, the flavanones, and the flavonols, respectively, was studied in an in vitro mixed culture model of the human colonic microflora. Time- and concentration-dependent degradation of all three compounds was observed, which was associated with the following metabolic events after cleavage of the ester or glycosidic bond: reduction of the aliphatic double bond of the resulting hydroxycinnamate caffeic acid residue; dehydroxylation and ring fission of the heterocyclic C-ring of the resulting deglycosylated flavanone, naringenin, and of the deglycosylated flavonol, quercetin (which differed depending on the substitution). The metabolic events, their sequences, and major phenolic end products, as identified by GC-MS or LC-MS/MS, were elucidated from the structural characteristics of the investigated compounds. The major phenolic end products identified were 3-(3-hydroxyphenyl)-propionic acid for chlorogenic acid, 3-(4-hydroxyphenyl)-propionic acid and 3-phenylpropionic acid for naringin, and 3-hydroxyphenylacetic acid and 3-(3-hydroxyphenyl)-propionic acid for rutin. The degree of degradation of the compounds studied was significantly influenced by the substrate concentration as well as individual variations in the composition of the fecal flora. The results support extensive metabolism of dietary polyphenols in the colon, depending on substrate concentration and residence time, with resultant formation of simple phenolics, which can be considered biomarkers of colonic metabolism if subsequently absorbed. It is also apparent that a relatively small number of phenolic degradation products are formed in the colon from the diverse group of natural polyphenols.

The metabolic fate of dietary polyphenols in humans.

Dietary polyphenols are widely considered to contribute to health benefits in humans. However, little is yet known concerning their bioactive forms in vivo and the mechanisms by which they may contribute toward disease prevention. Although many studies are focusing on the bioavailability of polyphenols through studying their uptake and the excretion of their conjugated forms, few are emphasizing the occurrence of metabolites in vivo formed via degradation by the enzymes of colonic bacteria and subsequent absorption. The purpose of this research was to investigate the relationship between biomarkers of the colonic biotransformation of ingested dietary polyphenols and the absorbed conjugated polyphenols. The results show that the majority of the in vivo forms derive from cleavage products of the action of colonic bacterial enzymes and subsequent metabolism in the liver. Those include the glucuronides of 3-hydroxyphenylacetic, homovanillic, vanillic and isoferulic acid as well as 3-(3-methoxy-4-hydroxyphenyl)-propionic, 3-(3-hydroxyphenyl)-propionic acid, and 3-hydroxyhippuric acid. In contrast, intact conjugated polyphenols themselves, such as the glucuronides of quercetin, naringenin and ferulic, p-coumaric, and sinapic acid were detected at much lower levels. The results suggest that consideration should be given to the cleavage products as having a putative role as physiologically relevant bioactive components in vivo.

Uptake and metabolism of epicatechin and its access to the brain after oral ingestion.

Epicatechin is a flavan-3-ol that is commonly present in green teas, red wine, cocoa products, and many fruits, such as apples. There is considerable interest in the bioavailability of epicatechin after oral ingestion. In vivo studies have shown that low levels of epicatechin are absorbed and found in the circulation as glucuronides, methylated and sulfated forms. Recent research has demonstrated protective effects of epicatechin and one of its in vivo metabolites, 3'-O-methyl epicatechin, against neuronal cell death induced by oxidative stress. Thus, we are interested in the ability of ingested epicatechin to cross the blood brain barrier and target the brain. Rats were administered 100 mg/kg body weight/d epicatechin orally for 1, 5, and 10 d. Plasma and brain extracts were analyzed by HPLC with photodiode array detection and LC-MS/MS. This study reports the presence of the epicatechin glucuronide and 3'-O-methyl epicatechin glucuronide formed after oral ingestion in the rat brain tissue.

The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans.

Dietary nitrate is metabolized to nitrite by bacterial flora on the posterior surface of the tongue leading to increased salivary nitrite concentrations. In the acidic environment of the stomach, nitrite forms nitrous acid, a potent nitrating/nitrosating agent. The aim of this study was to examine the pharmacokinetics of dietary nitrate in relation to the formation of salivary, plasma, and urinary nitrite and nitrate in healthy subjects. A secondary aim was to determine whether dietary nitrate increases the formation of protein-bound 3-nitrotyrosine in plasma, and if dietary nitrate improves platelet function. The pharmacokinetic profile of urinary nitrate excretion indicates total clearance of consumed nitrate in a 24 h period. While urinary, salivary, and plasma nitrate concentrations increased between 4- and 7-fold, a significant increase in nitrite was only detected in saliva (7-fold). High dietary nitrate consumption does not cause a significant acute change in plasma concentrations of 3-nitrotyrosine or in platelet function.

Zinc-histidine complex protects cultured cortical neurons against oxidative stress-induced damage.

The levels of zinc in the brain are directly affected by dietary zinc and deficiency has been associated with alcohol withdrawal seizures, excitotoxicity, impaired learning and memory and an accelerated rate of dysfunction in aged brain. Although zinc is essential for a healthy nervous system, high concentrations of zinc are neurotoxic, thus it is important to identify the most effective forms of zinc for treatment of conditions of the central nervous system. Accumulating evidence suggests that zinc-histidine complex (Zn(His)(2)) has greater biological potency and enhanced bioavailability compared with other zinc salts and also has antioxidant potential. Therefore, in this study we investigated the ability of zinc-histidine to protect cultured cortical neurons against hydrogen peroxide-induced damage. Pre-treating neurons for 18 h with subtoxic concentrations of zinc-histidine (5-25 microM) improved neuronal viability and strongly inhibited hydrogen peroxide-induced (75 microM, 30 min) cell damage as assessed by MTT turnover and morphological analysis 24h later. Low concentrations of zinc-histidine were more neuroprotective than zinc chloride. There was evidence of an anti-apoptotic mechanism of action as zinc-histidine inhibited hydrogen peroxide-induced caspase-3 activation and c-jun-N-terminal kinase phosphorylation. In summary, zinc supplementation with zinc-histidine protects cultured neurons against oxidative insults and inhibits apoptosis which suggests that zinc-histidine may be beneficial in the treatment of diseases of the CNS associated with zinc deficiency.

The compositional characterisation and antioxidant activity of fresh juices from sicilian sweet orange (Citrus sinensis L. Osbeck) varieties.

Epidemiological evidence has suggested that consumption of fruit and vegetables reduces the risk of both cancer and cardiovascular diseases, potentially through the biological actions of components such as vitamin C, vitamin E, flavonoids and carotenoids. Citrus species are extremely rich sources in vitamin C and flavanones, a class of compounds which belongs to the flavonoids family. A comparison of the phenolic compositions, the ascorbic acid contents and the antioxidant activities of fresh Sicilian orange juices from pigmented (Moro, Tarocco and Sanguinello) and non-pigmented (Ovale, Valencia and Navel) varieties of orange (Citrus sinensis L. Osbeck), was undertaken. The simultaneous characterisation and quantification of the major flavanone, anthocyanin and hydroxycinnamate components were attained by HPLC with diode array detection. Differences between varieties in terms of the flavanone glycoside content, particularly hesperidin, were observed, with the Tarocco juices reporting the highest content. Furthermore, cyanidin-3-glucoside and cyanidin-3-(6"-malonyl)-glucoside were predominant in all the pigmented varieties, but their concentration was higher in the juices of the Moro variety. Quantitatively, the major antioxidant component of all juices was ascorbic acid and its concentration was significantly correlated (r = 0.74, P < 0.001) with the total antioxidant activity of the juices, determined in vitro using the ABTS radical cation decolorization assay. Similarly, hydroxycinnamates (r = 0.73, P < 0.01) and anthocyanins (r = 0.98, P < 0.001) content showed a good correlation with the determined antioxidant capacity. Therefore orange juices, particularly those rich in anthocyanins, may represent a significant dietary source of flavonoids.

Gender differences in steady-state levels of oxidative damage to DNA in healthy individuals.

Oxidative damage to DNA has often been used as a biomarker for oxidative stress and more specifically for cancer risk. Indeed, the measurement of oxidative damage to DNA, particularly of 8-hydroxyguanine (8OHG) and 8-hydroxy-2'-deoxyguanosine (8OHdG), has been adopted as a method for establishing the effects of antioxidant supplementation towards protection from certain cancers, cardiovascular and neuro-degenerative diseases, both in patients and healthy individuals. However, reported levels of 8OHdG or 8OHG vary considerably, possibly due to the different methodologies used, and only few data are available for the non-smoking and the female population. In this paper, steady-state levels of oxidative damage to DNA measured in a group of 20 males and 19 females are reported. Significant gender differences in levels of modified DNA bases such as 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine), 8-hydroxyadenine (8OHA) and 5-hydroxycytosine (5OHC), measured by gas chromatography-mass spectrometry (GC/MS), were observed. The results are discussed in relation to the Vitamin C and iron status of the subjects and to the existing, yet limited, literature data. The role of gender in predisposition to oxidative damage to DNA needs to be addressed in future studies.

The antioxidant activity of regularly consumed fruit and vegetables reflects their phenolic and vitamin C composition.

Recent studies are emphasising the importance and putative modes of action of specific flavonoids as bioactive components of the diet in in vivo and in vitro models. Thus, it is important to have a clear idea of the major phenolic families of which fruit and vegetables are comprised and the levels contained therein. Regularly consumed fruit and vegetables of mixed varieties available on the UK market were analysed for the composition of the major individual phenolic components. The total phenolic content (applying the Folin assay) and the vitamin C levels were also determined. The antioxidant capacities of aqueous/methanolic extracts were comparatively assessed using the TEAC (Trolox Equivalent Antioxidant Capacity), the FRAP (Ferric Reducing Ability of Plasma) and ORAC (Oxygen Radical Absorbance Capacity) assays, which comprise contributions from polyphenols, simple phenols and the ascorbate component. The results were calculated in terms of 100 g fresh weight (FW) uncooked portion sizes. Fruit and vegetables rich in anthocyanins (e.g. strawberry, raspberry and red plum) demonstrated the highest antioxidant activities, followed by those rich in flavanones (e.g. orange and grapefruit) and flavonols (e.g. onion, leek, spinach and green cabbage), while the hydroxycinnamate-rich fruit (e.g. apple, tomato, pear and peach) consistently elicited the lower antioxidant activities. The TEAC, FRAP and ORAC values for each extract were relatively similar and well-correlated with the total phenolic and vitamin C contents. The antioxidant activities (TEAC) in terms of 100 g FW uncooked portion size were in the order: strawberry>> raspberry = red plum >> red cabbage >>>grapefruit = orange > spinach > broccoli > green grape approximately/= onion > green cabbage > pea > apple > cauliflower tomato approximately/= peach=leek > banana approximately/= lettuce.

The metabolism of dietary polyphenols and the relevance to circulating levels of conjugated metabolites.

Berry extracts rich in anthocyanins have been linked to protective effects including the modulation of age-related neurological dysfunction and the improvement of the resistance of red blood cells against oxidative stress in vitro. In this study the bioavailability, metabolism and elimination of polyphenols from blackcurrant juice, rich in anthocyanins, flavonols, and hydroxycinnamates, were investigated. The four major native anthocyanidin glycosides of blackcurrant juice, delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside and cyanidin-3-rutinoside, were detected and identified in low amounts by HPLC and LC-MS in plasma and urine post-ingestion. Elimination of the anthocyanins was fast (maximum excretion after 1 h) and plasma levels (0-128.6 nmol/l) and total urinary exretion (0.07-1.35 mg; 0.007-0.133% of the dose ingested) were low. Most significantly, of the hydroxycinnamates, conjugated and free ferulic, isoferulic, p-coumaric, sinapic and vanillic acids were identified in plasma and urine, using GC-MS techniques. Quercetin and kaempferol (as glucuronides) and the proposed colonic metabolite of quercetin, 3-hydroxyphenylacetic acid, were detectable in a minority of subjects. Increased daily urinary hippuric, 4-hydroxyhippuric and 3-hydroxyhippuric acid levels were also observed post-ingestion in all volunteers.

Hesperetin glucuronide, a photoprotective agent arising from flavonoid metabolism in human skin fibroblasts.

There is considerable interest in the biological properties of flavonoids in terms of their antioxidant and cytoprotective actions. The interaction of the flavanone hesperetin with human skin fibroblasts (FEK4) has revealed the potential for metabolism to hesperetin glucuronide and its subsequent extrusion. As a consequence of this observation, the effectiveness of hesperetin glucuronides, in comparison with that of the aglycone form, in protecting against UV-A radiation has been investigated. The results indicate that hesperetin glucuronides, but not hesperetin, protect against UV-A-induced necrotic cell death.

Smoking has no effect on the amino acid composition of apolipoprotein B100 of LDL while directly influencing the antioxidant status.

Previous studies have demonstrated increased plasma levels of oxidised low-density lipoprotein (oxLDL) in chronic smokers, which has been associated with the extent of endothelial dysfunction. In this study we examine the relationship between the amino acid composition of apolipoprotein B100 (apo B) of low-density lipoprotein (LDL), by reverse phase HPLC after precolumn derivatisation, between smokers (> or =40 cigarettes/day) and nonsmokers in relation to their plasma and LDL antioxidant status. While there was a significant difference in the levels of plasma vitamin C and alpha-tocopherol between female smokers and nonsmokers, as well as in the levels of LDL alpha-tocopherol, there was no significant difference in the amino acid composition of apo B between the two groups.