The identification of flavonoids as glycosides in human plasma.

This study describes evidence for the absorption of flavonoids and their presence in human plasma in the glycosylated form by HPLC analysis with photodiode array detection. Rutin and other quercetin glycosides, phloridzin, as well as an anthocyanin are detected simultaneously. In addition, a compound eluting with the spectral properties of the aurone family is identified. The results reveal that phloretin and quercetin are absorbed from the diet as glycosides. The polyphenols are detected in plasma from non-supplemented humans at individual levels in the range 0.5-1.6 microM.

Oxidised low density lipoproteins induce iron release from activated myoglobin.

Recent reports have detected the presence of iron in human atherosclerotic lesions [Biochem. J. 286 (1992) 901-905]. This study provides evidence for a biochemical mechanism whereby iron is released from myoglobin by low density lipoprotein (LDL) which has become oxidised by the ferryl myoglobin species. The haem destabilisation and iron release are inhibited by monohydroxamate compounds and desferrioxamine through their ability to inhibit the propagation of LDL oxidation. Thus, iron may derive from the myoglobin released from ruptured cells in the oxidising environment of the atherosclerotic lesion.

The interaction between ruptured erythrocytes and low-density lipoproteins.

Low-density lipoproteins (LDL) are oxidatively modified on interaction with haem proteins. The interaction of ruptured erythrocytes with LDL induces oxidative damage as detected by alterations in electrophoretic mobility and the peroxidation of the polyunsaturated fatty acyl chains. Difference spectroscopy reveals that the amplification of the oxidative process by the haem protein is related to the transition of the oxidation state of the haemoglobin in the erythrocyte lysate from the oxy [X-FeII-O2] to the ferryl [X-FeIV = O] form. The incorporation of the lipid-soluble antioxidant, butylated hydroxy toluene, at specific time points during the LDL-erythrocyte interaction prolongs the lag phase to oxidation and eliminates the oxy-to-ferryl conversion of the haemoglobin. The timescale of this haem conversion is related to the antioxidant status of the LDL.

Structure-antioxidant activity relationships of flavonoids and phenolic acids.

The recent explosion of interest in the bioactivity of the flavonoids of higher plants is due, at least in part, to the potential health benefits of these polyphenolic components of major dietary constituents. This review article discusses the biological properties of the flavonoids and focuses on the relationship between their antioxidant activity, as hydrogen donating free radical scavengers, and their chemical structures. This culminates in a proposed hierarchy of antioxidant activity in the aqueous phase. The cumulative findings concerning structure-antioxidant activity relationships in the lipophilic phase derive from studies on fatty acids, liposomes, and low-density lipoproteins; the factors underlying the influence of the different classes of polyphenols in enhancing their resistance to oxidation are discussed and support the contention that the partition coefficients of the flavonoids as well as their rates of reaction with the relevant radicals define the antioxidant activities in the lipophilic phase.

Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants.

In this study the antioxidant activities of the hydroxycinnamic acids, chlorogenic, caffeic, ferulic and p-coumaric, have been investigated in peroxidising lipid systems mediated by metmyoglobin. The results show that the order of effectiveness in increasing the resistance of LDL to peroxidation, in protecting LDL cholesterol from oxidation and preventing the oxidative modification of the LDL apoprotein B100 is caffeic = chlorogenic > ferulic > p-coumaric acid. Assessment of the rates of reaction of the hydroxycinnamates with ferrylmyoglobin, a product of the reductive decomposition of lipid hydroperoxides, reveals that the compounds are more effective as peroxyl radical scavengers than reductants of ferryl myoglobin in peroxidising LDL systems mediated by haem proteins.

Total antioxidant activity of low density lipoproteins and the relationship with alpha-tocopherol status.

A rapid method is described for measuring the antioxidant activity of low density lipoproteins. Studies were undertaken on individuals attending a hyperlipidaemia clinic, an unsupplemented group and a group after supplementation with 300 mg dl-alpha-tocopherol acetate for nine weeks. The results show a positive correlation between the antioxidant activity and alpha-tocopherol content of LDL in the supplemented group.

Influence of sialic acid content of low-density lipoprotein particles on endothelium-dependent vasorelaxation in rabbit aorta.

Low-density lipoprotein (LDL) concentration in plasma is an important predictor for atherosclerosis, and desialylated LDL has been proposed to be particularly atherogenic. Atherosclerosis is also associated with vascular endothelial dysfunction. We therefore wished to test the hypothesis that removal of sialic acid residues from LDL increases its ability to inhibit endothelium-dependent vasorelaxation. We studied vasorelaxant responses to acetylcholine (ACh) in isolated rabbit aortic rings as a model of endothelium-dependent relaxation, in the presence or absence of LDL treated either with saline or with neuraminidase, to cleave sialic acid residues. Vasorelaxant responses to ACh were inhibited by 300 microg protein per ml saline-treated LDL (E(max) 77.5+/-4.5 vs. 89.7+/-2.2% in the absence of LDL, P<0.05). This inhibitory effect was not altered by neuraminidase treatment of LDL. In contrast, 300 microg protein per ml LDL, either saline- or neuraminidase-treated, did not affect vasorelaxant responses to the endothelium-independent dilator sodium nitroprusside. We found no correlation between sialic acid content of saline-treated LDL and its ability to inhibit endothelium-dependent vasorelaxation, in rabbit aortic rings, at a concentration of 300 microg protein per ml. Our results therefore suggest that sialic acid content is not an important determinant of the effect of LDL on vascular endothelium-dependent relaxation.

Oxidisability of low density lipoproteins in patients with carotid or femoral artery atherosclerosis.

Oxidation of low density lipoprotein (LDL) is implicated in the pathogenesis of atherosclerosis. In this study the susceptibility to oxidation of LDL (from patients with atherosclerosis) is related to the progression of the disease. LDL were isolated from 37 patients with demonstrable atherosclerotic plaques. The susceptibility of LDL to oxidation (induced by an exogenous oxidative stress) was assessed by measuring the breakdown products of lipid peroxidation, the increased formation of conjugated dienes, and changes in surface charge of the apolipoprotein B (apo B). Progression of the atherosclerotic plaque was assessed by measuring the maximum velocity of blood through the narrowest portion of the vessel at inclusion and after one year. Twenty-nine of the 37 samples taken were found to have LDL that were partially oxidised, whereas 8 samples showed LDL whose state of oxidation was within the normal range. Progression of the atherosclerotic plaque occurred in 19 (66%) of the 29 patients whose lipoproteins were partially oxidised compared with only 2 (25%) of the 8 patients with normal lipoproteins (P = 0.055, Fisher's exact test). These data support an association between the progression of atherosclerotic plaques in carotid and femoral vessels and the susceptibility to oxidation of LDL.

Inhibition of copper-mediated low density lipoprotein peroxidation by quinoline and indolinone nitroxide radicals.

Quinoline and indolinone nitroxide radicals are known to be efficient scavengers of oxygen-centred (rate constants (k) between 10(3) and 10(5)/M/sec) and carbon-centred radicals (almost diffusion-controlled rate). In this study, the relative effects of these compounds in protecting low density lipoprotein (LDL) from oxidation induced by copper have been investigated. The extent of lipid peroxidation was assessed by monitoring the increased conjugated diene formation, the altered surface charge of the apolipoprotein B and the generation of aldehydic breakdown products of oxidized LDL. All the nitroxides inhibited LDL peroxidation in a concentration-dependent manner. The corresponding hydroxylamines of the nitroxides were also studied and were shown to inhibit lipid peroxidation to almost the same extent as the parent nitroxide. The data indicates that this class of nitroxide radicals (and their reduced hydroxylamine forms) are effective lipophilic antioxidants with the quinoline nitroxide being more efficient than the indolinone nitroxides.

The polyphenolic content of fruit and vegetables and their antioxidant activities. What does a serving constitute?

Analysis of the major flavone, flavonol, anthocyanidin and hydroxycinnamic acid constituents (and their glycosides) of onion, tomato, egg plant and apple has been undertaken and the antioxidant activities of the phenolic extracts determined. The major phenolic antioxidant components of egg plant are chlorogenic acid in the flesh and a delphinidin conjugate in the skin. In the case of apple, the major phenolic antioxidants detected are chlorogenic acid, procyanidins/catechin compounds, rutin and phloridzin. Quercetin glycosides are well-known to be the major phenolic components of onion. Assessment of the antioxidant activities of a serving of 100g fresh weight fruit, vegetable and comparison with previously reported findings for 150 ml beverage (500 ml portion in the case of beer), expressed in micromol Trolox equivalents show that the antioxidant activities of 1 glass (150 ml) red wine equivalent to 12 glasses white wine equivalent to 2 cups of tea equivalent to 4 apples equivalent to 5 portions of onion equivalent to 5.5 portions egg plant equivalent to 3.5 glasses of blackcurrant juice equivalent to 3.5 (500 ml) glasses of beer equivalent to 7 glasses of orange juice equivalent to 20 glasses of apple juice (long life).

Absorption of ferulic acid from low-alcohol beer.

Flavonoids and monophenolic compounds have been well-described over recent years for their properties as antioxidants and scavengers of reactive oxygen and nitrogen species. A number of epidemiological studies implicate a role for flavonoids in reducing the risk of coronary heart disease. In particular, the focus has been on flavonol-rich fruit and vegetables and flavonoid-rich beverages, especially tea and red wine. Mechanisms of protection are unclear since the absorption, distribution, metabolism and elimination of dietary phenolics have not yet been extensively investigated. Here we report the bioavailability of ferulic acid, 4-hydroxy-3-methoxy-cinnamic acid, the major hydroxycinnamate in beer. Studies of the pharmacokinetics of urinary excretion of ferulic acid from low alcohol beer consumption in humans have been undertaken. The results show that ferulic acid is absorbed with a peak time for maximal excretion of ca. 8 h and the mean cumulative amount excreted is 5.8 +/- 3.2 mg. These findings are consistent with the uptake of ferulic acid from dietary sources, such as tomatoes, and suggest that ferulic acid is more bioavailable than individual dietary flavonoids and phenolics so far studied.

Comparison of the phenolic composition of fruit juices by single step gradient HPLC analysis of multiple components versus multiple chromatographic runs optimised for individual families.

After minimal sample preparation, two different HPLC methodologies, one based on a single gradient reversed-phase HPLC step, the other on multiple HPLC runs each optimised for specific components, were used to investigate the composition of flavonoids and phenolic acids in apple and tomato juices. The principal components in apple juice were identified as chlorogenic acid, phloridzin, caffeic acid and p-coumaric acid. Tomato juice was found to contain chlorogenic acid, caffeic acid, p-coumaric acid, naringenin and rutin. The quantitative estimates of the levels of these compounds, obtained with the two HPLC procedures, were very similar, demonstrating that either method can be used to analyse accurately the phenolic components of apple and tomato juices. Chlorogenic acid in tomato juice was the only component not fully resolved in the single run study and the multiple run analysis prior to enzyme treatment. The single run system of analysis is recommended for the initial investigation of plant phenolics and the multiple run approach for analyses where chromatographic resolution requires improvement.

Isomerization of dietary lycopene during assimilation and transport in plasma.

Diets of individuals were supplemented with tomatoes, either cooked or as tomato pureé in order to compare uptake of lycopene from intact and homogenized fruit tissue matrices. Following a diet containing cooked tomatoes over three consecutive 7-day periods, little change in the carotenoid levels in plasma lipoproteins occurred. In contrast, a diet supplemented with concentrated tomato pureé, over a 2 week period, caused a significant (p < 0.05) increase in lycopene levels in plasma, showing that the lycopene within intact cells is less bioavailable than that from processed tissue. The isomeric composition of plasma lycopene was significantly different to that of the ingested pureé. A number of cis-isomers (predominantly 5-cis, 13-cis and 9-cis-) were detected in plasma, that are not present in the lycopene from pureé. The significance of the increase in lycopene following dietary supplementation with respect to bioavailability and the causes of isomerization are discussed.

Levels of antioxidant nutrients in plasma and low density lipoproteins: a human volunteer supplementation study.

A human supplementation study was undertaken in order to investigate the correlation between the intake of individual daily dosages of vitamin E (300 mg), vitamin C (250 mg), or beta-carotene (15 mg) of eight week duration and their uptake in vivo in plasma and LDL. The effects of a combined supplement of vitamin E, vitamin C and beta-carotene (Redoxon protector-75 mg, 150 mg, 15 mg respectively) were also investigated. The results show that on supplementation with the individual antioxidants the increases in plasma alpha-tocopherol:cholesterol levels lie in the 1.5-2 fold range and the beta-carotene:cholesterol ratios give a mean 3.5 fold enhancement. The combined supplement containing the same level of beta-carotene as the single dosage achieved comparative levels of uptake in plasma. The level of plasma vitamin C appears to be maximal at about 100 microM regardless of the pre-supplementation level.

Reference values for alpha-tocopherol and beta-carotene in the Whitehall II Study.

Plasma alpha-tocopherol, beta-carotene, serum lipids and their derived ratios were determined in British Civil Servants (n = 7177) at the second medical examination of the Whitehall II Study, a longitudinal study of cardiovascular disease. For plasma alpha-tocopherol the non-parametric 95% reference interval (90% confidence limits) for the total population was: 11.1 (10.9-11.3)-51.5 (50.6-52.7) mumol/l. For plasma beta-carotene the non-parametric reference interval for the total population was: 0.05 (0.05-0.05)-2.14 (2.08-2.21) mumol/l. The latter interval was wider than those previously published with a higher mean (0.61 mumol/l) and median (0.75 mumol/l). Plasma beta-carotene concentrations were higher in women than men with age-adjusted means of 0.70 and 0.57 mumol/l respectively (p < 0.001). This may reflect differences in diet, lifestyle and metabolism between the sexes. The alpha-tocopherol/cholesterol ratio, as in other surveys, did not vary with age. Among men, current- and ex-smokers had a higher alpha-tocopherol/cholesterol ratio than never-smokers with age-adjusted means of 4.18, 4.19 mumol/mmol and 4.05 mumol/mmol respectively. This difference is as yet unexplained. Follow-up of these subjects will help to clarify the role of antioxidant nutrients as protective factors for cardiovascular disease and cancer.

Antioxidant activity of phytoestrogenic isoflavones.

The aim of this work was to determine the antioxidant activities of a range of phytoestrogenic isoflavones. The antioxidant activity in the aqueous phase was determined by means of the ABTS.+ total antioxidant activity assay. The results show that the order of reactivity in scavenging the radical in the aqueous phase is genistein > daidzein = genistin approximately equal to biochanin A = daidzin > formononetin approximately equal to ononin, the latter displaying no antioxidant activity. The importance of the single 4'-hydroxyl group in the reactivity of the isoflavones, as scavengers of aqueous phase radicals, as well as the 5'7-dihydroxy structure is demonstrated. Examination of their abilities to enhance the resistance of low density lipoproteins to oxidation supports the observation that genistein is the most potent antioxidant among this family of compound studied, both in the aqueous and in the lipophilic phases.

Protective actions of tamoxifen and 4-hydroxytamoxifen against oxidative damage to human low-density lipoproteins: a mechanism accounting for the cardioprotective action of tamoxifen?

Tamoxifen and 4-hydroxytamoxifen protect isolated human low-density lipoproteins (LDLs) against copper-ion-dependent lipid peroxidation: 4-hydroxytamoxifen is more protective than tamoxifen or 17 beta-oestradiol. 4-Hydroxytamoxifen and 17 beta-oestradiol also prevent the increase in the electrophoretic mobility of LDL caused by exposure to copper ions, presumably by protection of the apoprotein B of LDL against oxidative modification. Our observations may help to account for the cardioprotective benefits reported to be associated with tamoxifen therapy and prophylaxis in breast cancer.

Inhibition of nitrous acid-dependent tyrosine nitration and DNA base deamination by flavonoids and other phenolic compounds.

Exposure of tyrosine or DNA bases to acidic nitrite at low pH results in the nitration of tyrosine and the formation of base deamination products, respectively. At pH 1, hypoxanthine and xanthine are formed from the deamination of adenine and guanine, respectively, whereas under the same conditions, uracil is not detected. The yield of 3-nitrotyrosine derived from interaction of equimolar nitrite and tyrosine at pH 1 is approximately 50% of that obtained from equimolar peroxynitrite-tyrosine interactions at pH 7. 4. The ability of a range of plant phenolic constituents to prevent damage mediated by acidic nitrite was also examined in comparison with the activity of vitamin C. The epicatechin/gallate family of flavonols, constituents of green tea, red wine, etc., demonstrates the most extensive inhibitory properties against both tyrosine nitration and base deamination. The results also show that ascorbic acid is a poor inhibitor of nitration or deamination under acidic conditions such as those of the stomach. The ability of plant phenolics to scavenge reactive nitrogen species derived from acidic nitrite may contribute to the protective effects of tea polyphenols against gastric cancer.