George Paganga’s research while affiliated with King's College London and other places

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.

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.

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.

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.

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.

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).

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.

1. Dietary supplementation with vitamin E reduces ischaemic events in patients with established coronary artery disease and improves endothelial function in cholesterol-fed rabbits. We examined whether such dietary supplementation with vitamin E improves endothelial function in patients with mild hypercholesterolaemia and coronary artery disease. 2. Twenty patients (total cholesterol 6.8 ± 1.1 mmol/l, mean ± SD) with angiographically documented coronary artery disease were randomly allocated to receive placebo (n = 10) or vitamin E, 400 i.u. daily, (n = 10) for 8 weeks. Endothelium-dependent and independent vasodilatation within forearm vasculature was assessed by brachial artery infusion of acetylcholine (co-infused with saline vehicle and l-arginine) and nitroprusside before and after supplementation. 3. Plasma concentrations of vitamin E increased from 32.9 ± 3.8 to 69.1 ± 11.8 μmol/l (means ± SE) in the vitamin E-supplemented group (P < 0.01) but did not change significantly in the placebo group. Lipid profiles remained similar before and after supplementation in both groups. Forearm blood flow responses to acetylcholine (7.5 and 15 μg/min) and nitroprusside (3 and 10 μg/min) were similar before and after supplementation in both groups. Acute intra-arterial administration of l-arginine (10 mg/min) augmented the response to acetylcholine (15 μg/min) in both groups before and after supplementation to a similar degree (mean augmentation: 60 ± 18%, P < 0.01). 4. Acute administration of l-arginine reverses endothelial dysfunction in forearm vasculature of patients with mild hypercholesterolaemia and coronary artery disease but supplementation with vitamin E (400 i.u. daily) for 8 weeks does not reverse l-arginine-responsive endothelial dysfunction.

Oxidation of low-density lipoprotein (LDL) is of great interest for epidemiological and clinical diagnostic reasons, as well as for basic scientific research, because it is thought to precede the development of atherosclerotic lesions (1). Measurement of individual antioxidants such as α-tocopherol, is, of course, a primary route to the study of LDL oxidation. However, such measurements are not always possible and do not take into account the potential contribution to the antioxidant activity of LDL from a wide variety of different antioxidants whose exact nature may not be known. Oxidized LDL can itself be quantitatively measured (2) as well as LDL oxidizability (3–5). However, the measurement of LDL oxidizability involves a prolonged and variable incubation period and the results obtained from the studies previously quoted are, in a number of respects, contradictory. LDL oxidizability measurements clearly reflect a number of factors other than the antioxidant content of LDL (for example, the presence of pre-formed lipid hydroperoxides and the ratio of saturated to unsaturated fatty acids) (6), which may, of course, be a desirable feature because oxidized LDL itself is the end product of a multi-factorial process.

Plasma α-tocopherol, β-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 α-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) μmol/1. For plasma p-carotene the nonparametric reference interval for the total population was: 0.05 (0.05–0.05) - 2.14 (2.08–2.21) μmol/1. The latter interval was wider than those previously published with a higher mean (0.61 μmol/1) and median (0.75 μmol/1). Plasma β-carotene concentrations were higher in women than men with age-adjusted means of 0.70 and 0.57 μmol/1 respectively (p i 0.001). This may reflect differences in diet, lifestyle and metabolism between the sexes. The a-tocopherol/cholesterol ratio, as in other surveys, did not vary with age. Among men, current- and ex-smokers had a higher α-tocopheroll cholesterol ratio than never-smokers with age-adjusted means of 4.18, 4.19 μmol/mmol and 4.05 μmol/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.