Anna R Proteggente’s research while affiliated with University of Surrey and other places

We have compared the biokinetics of deuterated natural (RRR) and synthetic (all rac) alpha-tocopherol in male apoE4-carrying smokers and nonsmokers. In a randomized, crossover study subjects underwent two 4-week treatments (400 mg/day) with undeuterated RRR- and all rac-alpha-tocopheryl acetate around a 12-week washout. Before and after each supplementation period subjects underwent a biokinetic protocol (48 h) with 150 mg deuterated RRR- or all rac-alpha-tocopheryl acetate. During the biokinetic protocols, the elimination of endogenous plasma alpha-tocopherol was significantly faster in smokers (P < 0.05). However, smokers had a lower uptake of deuterated RRR than nonsmokers, but there was no difference in uptake of deuterated all rac. The supplementation regimes significantly raised plasma alpha-tocopherol (P < 0.001) with no differences in response between smokers and nonsmokers or between alpha-tocopherol forms. Smokers had significantly lower excretion of alpha-carboxyethyl-hydroxychroman than nonsmokers following supplementation (P < 0.05). Nonsmokers excreted more alpha-carboxyethyl-hydroxychroman following RRR than all rac; however, smokers did not differ in excretion between forms. At baseline, smokers had significantly lower ascorbate (P < 0.01) and higher F(2)-isoprostanes (P < 0.05). F(2)-isoprostanes in smokers remained unchanged during the study, but increased in nonsmokers following alpha-tocopherol supplementation. These data suggest that apoE4-carrying smokers and nonsmokers differ in their handling of natural and synthetic alpha-tocopherol.

The deleterious impact of cigarette smoking on cardiovascular health may be in part attributable to a free radical mediated proinflammatory response in circulating monocytes. In the current investigation, the impact of vitamin C supplementation on monocyte gene expression was determined in apoE4 smokers versus non-smokers. A total of 10 smokers and 11 non-smokers consumed 60mg/day of vitamin C for four weeks and a fasting blood sample was taken at baseline and post-intervention for the determination of plasma vitamin C and monocyte gene expression profiles using cDNA array and real time PCR. In apoE4 smokers, supplementation resulted in a 43% increase in plasma vitamin C concentrations. Furthermore, a number of genes were differentially expressed more than 2-fold in response to treatment, including a downregulation of the proinflammatory mediators tumor necrosis factor (TNF) beta, TNF receptor, neurotrophin-3 growth factor receptor, and monocyte chemoattractant protein 1 receptor. The study has identified a number of molecular mechanisms underlying the benefit of vitamin C supplementation in smokers.

Previous studies comparing the biokinetics of deuterated natural (RRR) and synthetic (all-rac) alpha-tocopherol (vitamin E) used a simultaneous ingestion or competitive uptake approach and reported relative bioavailability ratios close to 2:1, higher than the accepted biopotency ratio of 1.36:1. The aim of the current study was to compare the biokinetics of deuterated natural and synthetic vitamin E using a noncompetitive uptake model both before and after vitamin E supplementation in a distinct population. Healthy men (n = 10) carrying the apolipoprotein (apo)E4 genotype completed a randomized crossover study, comprised of two 4-wk treatments with 400 mg/d (RRR-alpha-tocopheryl and all-rac-alpha-tocopheryl acetate) with a 12-wk washout period between treatments. Before and after each treatment period, the subjects consumed a capsule containing 150 mg deuterated alpha-tocopheryl acetate in either the RRR or all-rac form depending on their treatment regimen. Blood was obtained up to 48 h after ingestion, and tocopherols analyzed by LC/MS. After deuterated all-rac administration, plasma deuterated tocopherol maximum concentrations and area under the concentration vs. time curves (AUC) were lower than those following RRR administration. The RRR:all-rac ratios determined from the deuterated biokinetic profiles (maximum concentration; C(max)) and AUCs were 1.35:1 +/- 0.17 and 1.33:1 +/- 0.18, respectively. The 4-wk supplementation with either RRR or all-rac significantly increased plasma alpha-tocopherol concentrations (P < 0.001), but decreased the plasma response to newly absorbed deuterated RRR or all-rac alpha-tocopherol. Using a noncompetitive uptake approach, the relative bioavailability of natural to synthetic vitamin E in apoE4 males was close to the currently accepted biopotency ratio of 1.36:1.

Limited information is available on factors that can influence vitamin E bioavailability. In several studies we have investigated the influence of dietary, biochemical, and genetic factors on vitamin E biokinetics. In these studies, subjects ingested a capsule containing 150 mg deuterated RRR-alpha-tocopheryl acetate, blood was taken up to 48 hr, and tocopherols were analyzed by liquid chromatography and mass spectroscopy. There was significantly greater plasma-labeled alpha-tocopherol concentrations when the capsule was consumed with a high-fat meal (17.5 g) versus a low-fat meal (2.7 g), and there was also a difference between a high-fat toast and butter and a cereal with full-fat milk meal (both 17.5 g fat), indicating that both the amount of fat and food matrix is important for vitamin E absorption. Dyslipidemic subjects displayed a reduced plasma uptake of newly absorbed alpha-tocopherol, and differences were also apparent in individual lipoproteins. A decreased uptake of labeled alpha-tocopherol was also observed in erythrocytes, platelets, and lymphocytes of dyslipidemics. Following vitamin E supplementation (400 mg/day, 4 weeks), the uptake of newly absorbed alpha-tocopherol was decreased, presumably because of saturation of alpha-tocopherol transfer protein. We also found that apoE3 subjects displayed a considerably reduced uptake of newly absorbed labeled alpha-tocopherol compared to apoE4 subjects, which may be a consequence of the reduced low-density lipoprotein catabolic rate in these subjects. Taken together, these data show that several physiological factors influence the uptake of newly absorbed alpha-tocopherol, and that this is an important consideration in the design of future vitamin E supplementation studies.

Cigarette smoking is associated with increased oxidative stress and increased risk of degenerative disease. As the major lipophilic antioxidant, requirements for vitamin E may be higher in smokers due to increased utilisation. In this observational study we have compared vitamin E status in smokers and non-smokers using a holistic approach by measuring plasma, erythrocyte, lymphocyte and platelet alpha- and gamma-tocopherol, as well as the specific urinary vitamin E metabolites alpha- and gamma-carboxyethyl-hydroxychroman (CEHC). Fifteen smokers (average age 27 years, smoking time 7.5 years) and non-smokers of comparable age, gender and body mass index (BMI) were recruited. Subjects completed a 7-day food diary and on the final day they provided a 24 h urine collection and a 20 ml blood sample for measurement of urinary vitamin E metabolites and total vitamin E in blood components, respectively. No significant differences were found between plasma and erythrocyte alpha- and gamma-tocopherol in smokers and non-smokers. However, smokers had significantly lower alpha-tocopherol (mean+/-SD, 1.34+/-0.31 micromol/g protein compared with 1.94+/-0.54, P = 0.001) and gamma-tocopherol (0.19+/-0.04 micromol/g protein compared with 0.26+/-0.08, P = 0.026) levels in their lymphocytes, as well as significantly lower alpha-tocopherol levels in platelets (1.09+/-0.49 micromol/g protein compared with 1.60+/-0.55, P = 0.014; gamma-tocopherol levels were similar). Interestingly smokers also had significantly higher excretion of the urinary gamma-tocopherol metabolite, gamma-CEHC (0.49+/-0.25mg/g creatinine compared with 0.32+/-0.16, P = 0.036) compared to non-smokers, while their alpha-CEHC (metabolite of alpha-tocopherol) levels were similar. There was no significant difference between plasma ascorbate, urate and F2-isoprostane levels. Therefore in this population of cigarette smokers (mean age 27 years, mean smoking duration 7.5 years), alterations to vitamin E status can be observed even without the more characteristic changes to ascorbate and F2-isoprostanes. We suggest that the measurement of lymphocyte and platelet vitamin E may represent a valuable biomarker of vitamin E status in relation to oxidative stress conditions.

Cigarette smoking is associated with increased oxidative stress and increased risk of degenerative disease. As the major lipophilic antioxidant, requirements for vitamin E may be higher in smokers due to increased utilisation. In this observational study we have compared vitamin E status in smokers and non-smokers using a holistic approach by measuring plasma, erythrocyte, lymphocyte and platelet α- and γ-tocopherol, as well as the specific urinary vitamin E metabolites α- and γ-carboxyethyl-hydroxychroman (CEHC). Fifteen smokers (average age 27 years, smoking time 7.5 years) and non-smokers of comparable age, gender and body mass index (BMI) were recruited. Subjects completed a 7-day food diary and on the final day they provided a 24 h urine collection and a 20 ml blood sample for measurement of urinary vitamin E metabolites and total vitamin E in blood components, respectively. No significant differences were found between plasma and erythrocyte α- and γ-tocopherol in smokers and non-smokers. However, smokers had significantly lower α-tocopherol (mean±SD, 1.34±0.31 μmol/g protein compared with 1.94±0.54, P=0.001) and γ-tocopherol (0.19±0.04 μmol/g protein compared with 0.26±0.08, P=0.026) levels in their lymphocytes, as well as significantly lower α-tocopherol levels in platelets (1.09±0.49 μmol/g protein compared with 1.60±0.55, P=0.014; γ-tocopherol levels were similar). Interestingly smokers also had significantly higher excretion of the urinary γ-tocopherol metabolite, γ-CEHC (0.49±0.25 mg/g creatinine compared with 0.32±0.16, P=0.036) compared to non-smokers, while their α-CEHC (metabolite of α-tocopherol) levels were similar. There was no significant difference between plasma ascorbate, urate and F2-isoprostane levels. Therefore in this population of cigarette smokers (mean age 27 years, mean smoking duration 7.5 years), alterations to vitamin E status can be observed even without the more characteristic changes to ascorbate and F2-isoprostanes. We suggest that the measurement of lymphocyte and platelet vitamin E may represent a valuable biomarker of vitamin E status in relation to oxidative stress conditions.

To investigate the aetiology of chronic idiopathic axonal polyneuropathy (CIAP), 50 consecutive patients were compared with 50 control subjects from the same region. There were 22 patients with painful neuropathy and 28 without pain, 26 with sensory neuropathy and 24 with sensory and motor neuropathy. The typical picture was a gradually progressive sensory or sensory and motor neuropathy. It caused mild or sometimes moderate disability, and reduced the quality of life. There was no evidence that alcohol, venous insufficiency, arterial disease or antibodies to peripheral nerve antigens played a significant part. There was a possible history of peripheral neuropathy in the first or second-degree relatives of six patients and no controls (P = 0.01), and claw toes were present in 12 patients and four controls (P = 0.03). Thirty-two per cent of the patients and 14% of the controls had impaired glucose tolerance or fasting hyperglycaemia but, after adjusting for age and sex, the difference was not significant (P = 0.45), even in the painful neuropathy subgroup. The mean (SD) fasting insulin concentrations were significantly (P = 0.01) higher in the patients [75.9 (44.4) mmol/l] than the controls [47.3 (37.9) mmol/l], and the mean was higher still in the painful neuropathy subgroup [92.2 (37.1) mmol/l] (P < 0.0001). However, insulin resistance as assessed using the homeostasis model assessment formula was not significantly greater in the patients, even in those with pain, than the controls. After adjustment for body mass index as well as age and sex, there was no significant difference in the serum cholesterol concentrations, but there were significantly higher triglyceride concentrations in the patients [mean 1.90 (1.41) mmol/l] than the controls [mean 1.25 (0.79] mmol/l) (P = 0.02). In the patients with painful peripheral neuropathy, the mean triglyceride concentration was 2.37 (1.72), which was even more significantly greater compared with the controls (P = 0.003). In conclusion, CIAP is a heterogeneous condition. A logistic regression analysis identified environmental toxin exposure and hypertriglyceridaemia, but not glucose intolerance or alcohol overuse as significant risk factors that deserve further investigation as possible causes of CIAP.

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.

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.

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity.