Salka E. Nielsen

Effect of parsley ( Petroselinum crispum ) intake on urinary apigenin excretion, blood antioxidant enzymes and biomarkers for oxidative stress in human subjects

Seven men and seven women participated in a randomized crossover trial to study the effect of intake of parsley (Petroselinum crispum), containing high levels of the flavone apigenin, on the urinary excretion of flavones and on biomarkers for oxidative stress. The subjects received a strictly controlled diet low in flavones and other naturally occurring antioxidants during the 2 weeks of intervention. This basic diet was supplemented with parsley providing 3.73-4.49 mg apigenin/MJ in one of the intervention weeks. Urinary excretion of apigenin was 1.59-409.09 micrograms/MJ per 24 h during intervention with parsley and 0-112.27 micrograms/MJ per 24 h on the basic diet (P < 0.05). The fraction of apigenin intake excreted in the urine was 0.58 (SE 0.16)% during parsley intervention. Erythrocyte glutathione reductase (EC 1.6.4.1; GR) and superoxide dismutase (EC 1.15.1.1; SOD) activities increased during intervention with parsley (P < 0.005) as compared with the levels on the basic diet, whereas erythrocyte catalase (EC 1.11.1.6) and glutathione peroxidase (EC 1.11.1.9) activities did not change. No significant changes were observed in plasma protein 2-adipic semialdehyde residues, a biomarker of plasma protein oxidation. In this short-term investigation, an overall decreasing trend in the activity of antioxidant enzymes was observed during the 2-week study. The decreased activity of SOD was strongly correlated at the individual level with an increased oxidative damage to plasma proteins. However, the intervention with parsley seemed, partly, to overcome this decrease and resulted in increased levels of GR and SOD.

Effect of red wine and red grape extract on blood lipids, haemostatic factors, and other risk factors for cardiovascular disease

Objective:Some epidemiological studies found a lower risk of cardiovascular disease among wine drinkers than among drinkers of other types of ethanol. This difference might be due to an effect of nonalcohol compounds in wine on important cardiovascular risk factors. The objective of this study was to compare the effect of red wine, nonalcohol compounds of red wine and placebo on established cardiovascular risk factors.Design:A parallel, four-armed intervention study.Subjects:A total of 69 healthy 38–74-y-old men and women.Interventions:Subjects were randomised to either 1: red wine (males: 300 ml/day, 38.3 g alcohol/day, female subjects: 200 ml/day, 25.5 g alcohol/day), 2: water+red grape extract tablets (wine-equivalent dose), 3: water+red grape extract tablets (half dose), or 4: water+placebo tablets for a period of 4 weeks. No other sources of alcohol or anthocyanin were allowed. Plasma high-density lipoprotein (HDL)-cholesterol (HDL-C), low-density lipoprotein (LDL)-cholesterol (LDL-C), HDL-C/LDL-C-ratio, very-low-density lipoprotein (VLDL)-triacylglycerol, total cholesterol, fibrinogen, factor VII coagulant activity (FVIIc), blood pressure, and body weight were determined before and after intervention.Results:Wine consumption was associated with a significant 11–16% increase in fasting HDL-C and 8–15% decrease in fasting fibrinogen relative to not drinking wine. There were no significant treatment effects on fasting LDL-C, HDL-C/LDL-C-ratio, VLDL-triacylglycerol, total cholesterol, FVIIc, or blood pressure. Drinking wine was associated with relative body weight increments closely corresponding to the energy contributed by the alcohol component.Conclusion:Moderate red wine consumption for 4 weeks is associated with desirable changes in HDL-C and fibrinogen compared with drinking water with or without red grape extract. The impact of wine on the measured cardiovascular risk factors thus seems primarily explained by an alcohol effect. Our finding suggests that the putative difference in cardiac risk associated with wine vs other alcoholic beverages might be rather explained by other life-style confounders than by red wine contents of nonalcohol components.Sponsorship:This study was supported by Chr. Hansen A/S, Denmark.

Green tea extract only affects markers of oxidative status postprandially: lasting antioxidant effect of flavonoid-free diet

Epidemiological studies suggest that foods rich in flavonoids might reduce the risk of cardiovascular disease and cancer. The objective of the present study was to investigate the effect of green tea extract (GTE) used as a food antioxidant on markers of oxidative status after dietary depletion of flavonoids and catechins. The study was designed as a 2 x 3 weeks blinded human cross-over intervention study (eight smokers, eight non-smokers) with GTE corresponding to a daily intake of 18.6 mg catechins/d. The GTE was incorporated into meat patties and consumed with a strictly controlled diet otherwise low in flavonoids. GTE intervention increased plasma antioxidant capacity from 1.35 to 1.56 (P<0.02) in postprandially collected plasma, most prominently in smokers. The intervention did not significantly affect markers in fasting blood samples, including plasma or haemoglobin protein oxidation, plasma oxidation lagtime, or activities of the erythrocyte superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase. Neither were fasting plasma triacylglycerol, cholesterol, alpha-tocopherol, retinol, beta-carotene, or ascorbic acid affected by intervention. Urinary 8-oxo-deoxyguanosine excretion was also unaffected. Catechins from the extract were excreted into urine with a half-life of less than 2 h in accordance with the short-term effects on plasma antioxidant capacity. Since no long-term effects of GTE were observed, the study essentially served as a fruit and vegetables depletion study. The overall effect of the 10-week period without dietary fruits and vegetables was a decrease in oxidative damage to DNA, blood proteins, and plasma lipids, concomitantly with marked changes in antioxidative defence.

Column-switching high-performance liquid chromatographic assay for the determination of quercetin in human urine with ultraviolet absorbance detection.

A high-performance liquid chromatographic method is described for the determination of quercetin in human urine using column-switching and ultraviolet (UV) absorbance detection. Urine samples were enzymatically hydrolysed and solid-phase extracted prior to injection onto the high-performance liquid chromatography (HPLC) system. Prior to elution of quercetin and the internal standard, fisetin. from the first column used for sample clean-up, the six-port valve was switched to the second column for analysis with UV detection. Detection of quercetin was accurate and reproducible, with a detection limit of 5 ng/ml. The method was applied to determine the urinary level of quercetin in 120 samples from an intervention study with fruit juice.

Effects of Commonly Consumed Fruit Juices and Carbohydrates on Redox Status and Anticancer Biomarkers in Female Rats

Administration of apple juice, black currant juice, or a 1:1 combination of the two juices significantly decreased the level of the lipid peroxidation biomarker malondialdehyde in plasma of female rats, whereas the protein oxidation biomarker 2-amino-adipic semialdehyde, was significantly increased following administration of orange juice, black currant juice, or the 1:1 combination of apple and black currant juice. A significant increase in 2-amino-adipic semialdehyde was also observed in control rats given sucrose, fructose, and glucose in the drinking water at concentrations approximating the average carbohydrate levels in the employed fruit juices. None of the fruit juices were found to affect the activities of antioxidant enzymes in red blood cells or hepatic glutathione S-transferase. Hepatic quinone reductase activity, on the other hand, was significantly increased by grapefruit juice, apple juice, and black currant juice. The total daily intake of a selected subset of flavonoid aglycones ranged from 0.2 to 4.3 mg, and quercetin was found to be a minor constituent of all the juices investigated. In a parallel study, rats were fed quercetin at doses ranging from 0.001 to 10 g/kg of diet. However, no effects were observed on hepatic glutathione S-transferase or quinone reductase activities, plasma redox status, or the activity of red blood cell antioxidant enzymes. Overall, the results of the present study suggest that commonly consumed fruit juices can alter lipid and protein oxidation biomarkers in the blood as well as hepatic quinone reductase activity, and that quercetin may not be the major active principle. The observation that natural carbohydrates are capable of mediating oxidative stress in vivo warrants further studies due to the central role refined and unrefined carbohydrates play in human nutrition.

Simultaneous determination of hydroxycinnamates and catechins in human urine samples by column switching liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry.

A quantitative liquid chromatography mass spectrometry (LC-MS) methodology with online sample clean up by column switching is described for the simultaneous determination of the hydroxycinnamates, caffeic acid and chlorogenic acid, and of the catechins, epicatechin and catechin in human urine samples. Enzymatically treated urine samples were directly injected onto the LC-MS system, where sample clean up was performed by a reversed-phase Zorbax 300SB C(3) column and selective elution of the target compounds onto a Zorbax SB C(18) column resulted in final separation prior to detection by atmospheric pressure chemical ionization (APCI) MS using single ion monitoring (SIM) in negative mode. Linear calibration graphs were achieved in the dynamic range of 10-1000 ng/ml urine. The inter- and intraassay coefficients of variation (C.V.%) for the analysis of the four compounds in quality control urine samples were between 7.8 and 10.9, n=17 (reproducibility), and the repeatability of the assay was between 2.5 and 5.0% (n=12). Analyses of urine samples from a human dietary intervention study with intake of 600 g of fruits and vegetables were demonstrated. To our knowledge, this is the first method described that allows simultaneous determination of both hydroxycinnamates and catechins in biological samples.

Triterpenoid saponins from Phytolacca rivinoides and Phytolacca bogotensis

Investigation of the ethanolic extracts from Phytolacca rivinoides and P. bogotensis has resulted in the isolation of five new triterpenoid glycosides of serjanic acid. Their structures have been established mainly by spectroscopic methods (FAB-MS, 1H, 13C NMR, COSY, NOESY, TOCSY, HETCOR and J-resolved 1H NMR) as 3-O-(O-beta-D-galactopyranosyl-(1-->3)-O-beta-D-glucopyranosyl)serjan ic acid 28-O-beta-D-glucopyranosyl ester, 3-O-(O-beta-D-glucopyranosyl-(1-->3)-O-[beta-D-galactopyranosyl-(1-->4)] -O- beta-D-glucopyranosyl)serjanic acid 28-O-beta-D-glucopyranosyl ester, 3-O-(O-alpha-L-rhamnopyranosyl-(1-->2)-O-beta-D-glucopyranosyl-(1-->2)- O-beta-glucopyranosyl)serjanic acid 28-O-beta-D-glucopyranosyl ester, 3-O-(O-beta-D-glucopyranosyl-(1-->3)-O-beta-D-galactopyranosyl-(1-->4)- O-beta-D-glucopyranosyl)serjanic acid 28-O-beta-D-glucopyranosyl ester and 3-O-(O-beta-D-galactopyranosyl-(1-->4)-O-[beta-D-glucopyranosyl-(1-->3)] - O-beta-D-glucopyranosyl)serjanic acid.

Immunological Methods for Dosimetry of Heterocyclic Amines

Carcinogenic heterocyclic amines are formed by common household cooking procedures, and it is therefore of interest to evaluate exposures in the general population. High-affinity monoclonal antibodies to 2-amino-l-methyl-6-phenyl- imidazo[4,5-b]pyridine (PhIP1) were produced and a sensitive fluorescence-based ELISA assay developed with a 50% inhibition by 150 fmoles PhIP or PhlP- metabolites modified in the 2-position. PhIP antigenic material was excreted mainly from 0-24 hours after a single meal containing fried meat in two volunteers and dropped to background levels after 48 hours. Urine samples (24 hours) were collected from 265 Danish males and females aged 40-70 years. The participants were also asked to fill in a questionnaire regarding their recent intake of fried and otherwise cooked meat. Excretion of PhlP-antigenic material was observed among those who had been eating Med meat with a median excretion of 1.5μg/24 hours. A higher median excretion among all samples of about 2.3μg/24 hours was observed when acid hydrolyzed samples were analyzed. This indicates that a large proportion of conjugated metabolites are present before hydrolysis, since these metabolites are not as efficient competitors for the antibody in ELISA. The excretion of PhIP antigenic materials showed a skewed normal distribution. These observations are close to predicted exposure rates in the general population, based on food consumption figures and analyses of PhIP in fried meat samples. PhIP antigenic material was always found to be present in urine samples from individuals who reported a recent intake of fried meat. Positive samples were also found among individuals who reported no recent intakes of fried or browned meat. The possible sources of this exposure are discussed.

EFFECT OF FRUIT JUICE INTAKE ON URINARY QUERCETIN EXCRETION AND BIOMARKERS OF ANTIOXIDATIVE STATUS

BACKGROUND Epidemiologic studies suggest that foods rich in flavonoids might reduce the risk of cardiovascular disease. OBJECTIVE Our objective was to investigate the effect of intake of flavonoid-containing black currant and apple juice on urinary excretion of quercetin and on markers of oxidative status. DESIGN This was a crossover study with 3 doses of juice (750, 1000, and 1500 mL) consumed for 1 wk by 4 women and 1 man corresponding to an intake of 4.8, 6.4, and 9.6 mg quercetin/d. RESULTS Urinary excretion of quercetin increased significantly with dose and with time. The fraction excreted in urine was 0.29-0.47%. Plasma quercetin did not change with juice intervention. Plasma ascorbate increased during intervention because of the ascorbate in the juice. Total plasma malondialdehyde decreased with time during the 1500-mL juice intervention, indicating reduced lipid oxidation in plasma. Plasma 2-amino-adipic semialdehyde residues increased with time and dose, indicating a prooxidant effect of the juice, whereas erythrocyte 2-aminoadipic semialdehyde and gamma-glutamyl semialdehyde concentrations, Trolox-equivalent antioxidant capacity, and ferric reducing ability of plasma did not change. Glutathione peroxidase activity increased significantly with juice dose. CONCLUSIONS Urinary excretion of quercetin seemed to be a small but constant function of quercetin intake. Short-term, high intake of black currant and apple juices had a prooxidant effect on plasma proteins and increased glutathione peroxidase activity, whereas lipid oxidation in plasma seemed to decrease. These effects might be related to several components of the juice and cannot be attributed solely to its quercetin content.