Evaluation of flavonoids and enterolactone in overnight urine as intake biomarkers of fruits, vegetables and beverages in the Inter99 cohort study using the method of triads.

Since collection of 24 h urine samples is very time consuming and difficult to obtain, epidemiological studies typically only obtain spot urine samples. The aim of the present study was to evaluate whether flavonoids and enterolactone in overnight urine could substitute flavonoids and enterolactone in 24 h urine as an alternative and more feasible biomarker of fruit, vegetable and beverage intake. A total of 191 individuals in the Inter99 cohort in Denmark completed the validation study. Concentrations of nine urinary flavonoid aglycones (quercetin, isorhamnetin, tamarixetin, kaempferol, hesperetin, naringenin, eriodictyol, phloretin and apigenin) and enterolactone were determined in overnight and 24 h urine samples, and their validity as biomarkers of fruit, vegetable and beverage intake was evaluated in relation to two independent reference methods (Inter99 FFQ data and plasma carotenoids) by using the method of triads. The intakes of fruit, juice, vegetables and tea reported in the FFQ were reflected by the flavonoid biomarker both in overnight and 24 h urine samples. Validity coefficients for the flavonoid biomarker in overnight urine ranged from 0·39 to 0·49, while the corresponding validity coefficients for the biomarker in 24 h urine ranged from 0·43 to 0·66. Although the validity coefficients were lower for overnight urine than for the 24 h urine flavonoid biomarker, they were still of acceptable magnitude. In conclusion, the results indicate that flavonoids and enterolactone in overnight urine samples may be used as a more feasible biomarker than 24 h urine for the assessment and validation of fruit, juice, vegetable and tea intakes in epidemiological studies.

Absorption, conjugation and excretion of the flavanones, naringenin and hesperetin from alpha-rhamnosidase-treated orange juice in human subjects.

We have determined the absorption, conjugation and excretion of naringenin-7-O-rutinoside (narirutin) compared to the corresponding glucoside in an orange juice matrix in human subjects. Healthy volunteers (eight men and eight women), in a double blind, randomised, crossover study, consumed orange juice with (1) natural content of naringenin-7-O-rutinoside; (2) alpha-rhamnosidase-treated to yield naringenin-7-O-glucoside. Blood was sampled at twelve time points and three fractions of urine were collected over 24 h. The area under the plasma-time curve of naringenin from (2) alpha-rhamnosidase-treated orange juice was increased about 4-fold (P < 0.0001), peak plasma concentration (Cmax) was 5.4-fold higher (P < 0.0001) and Tmax was decreased from 311 to 92 min (P = 0.002) compared to untreated orange juice (1), indicating a change in absorption site from the colon to the small intestine. Furthermore, the amount in urine was increased from 7 to 47 % (P < 0.0001) of the dose after consumption of the alpha-rhamnosidase-treated orange juice (2). All urine samples contained both naringenin-7- and -4'-O-glucuronides. In addition, to examine the effect of dose and alpha-rhamnosidase treatment on hesperetin conjugate profiles, a further treatment where (3) orange juice fortified with three times the original content of hesperetin-7-O-rutinoside was used. Five hesperetin metabolites (3'-O-glucuronide; 7-O-glucuronide; 5,7-O-diglucuronide; 3',7-O-diglucuronide; 3'-O-sulphate) were present after all treatments (1-3), with the same profile of the conjugates. The present data show that bioavailability of naringenin is increased by conversion from rutinoside to glucoside, but the profile of the conjugates of flavanones formed and excreted in urine is neither affected by the absorption site nor by a 3-fold change in dose.

Effects of red grape skin and seed extract supplementation on atherosclerosis in Watanabe heritable hyperlipidemic rabbits.

Epidemiological studies have suggested an association between consumption of red wine and other polyphenolic compounds and prevention of cardiovascular diseases. In the present study, Watanabe heritable hyperlipidemic (WHHL) rabbits were used to investigate the effects of polyphenols in a red grape skin and seed extract (GSE) on the development of atherosclerosis. WHHL rabbits received either semisynthetic diet (casein based) or semisynthetic diet added GSE over a period of 15 wk. Plasma lipids and aortic cholesterol accumulation were measured. Feeding semisynthetic diet was associated with increasing hypercholesterolemia, which was developing slower in GSE group compared to the controls as recorded by significantly lower plasma cholesterol in dosage week 7 (males: P < 0.05, females: P < 0.01) and 11 (males: P < 0.01). Aortic atherosclerosis evaluated as the cholesterol content in aortic tissue was comparable in the control and GSE-dosed females, but it was significantly reduced in the abdominal part of GSE-dosed male compared to the controls (P < 0.05). In conclusion, feeding GSE extract to WHHL rabbits had no significant effects in females but was associated with transient less hypercholesterolemic response to semisynthetic diet and, furthermore, retarded the development of aortic atherosclerosis in males as demonstrated by significantly lower cholesterol content in the abdominal part.

Improved synthesis methods of standards used for quantitative determination of total isothiocyanates from broccoli in human urine.

A well-known method for quantification of isothiocyanates (ITCs) and their metabolites is the condensation reaction with 1,2-benzenedithiole to produce 1,3-benzodithiole-2-thione, which can be quantified by high-performance liquid chromatography. Standards of an ITC metabolite and 1,3-benzodithiole-2-thione are required for this assay but are not commercially available. In the present study, we report on an improved synthesis of the ITC metabolite N-acetyl-S-(N-4-methylsulfinylbutylthiocarbamoyl)-L-cysteine and 1,3-benzodithiole-2-thione. The standards were used to quantify the urinary excretion of ITCs from 10 healthy subjects who consumed 350 g broccoli. The excretion was investigated throughout 48 h showing a cumulative urinary ITC excretion of 49.1+/-25.2% of the dose.

Self-reported dietary supplement use is confirmed by biological markers in the Norwegian Mother and Child Cohort Study (MoBa).

BACKGROUND/AIMS: A food frequency questionnaire (FFQ) and a database for dietary supplements were developed for use in the Norwegian Mother and Child Cohort Study (MoBa). The aim of the present study was to investigate the relation between reported use and biomarkers in supplement and non-supplement users and to validate self-reported intake of dietary supplements in mid pregnancy. METHOD: 120 women were recruited from MoBa, and 119 subjects completed the MoBa FFQ and a 4-day weighed food diary. Information on supplement use was collected by both methods. Venous blood specimens and 24-hour urine samples were obtained for measurement of dietary biomarkers. RESULTS: Biomarker concentration/excretion and intake differed significantly between supplement and non-supplement users for vitamin D, carotenoids, folate, the n-6/n-3 fatty acid ratio and iodine (p < 0.05 for all variables). Flavonoid excretion was higher in flavonoid-supplement users (p < 0.05). Significant correlations between total dietary intake (food and supplements) and biomarker concentration/excretion were found for vitamin D (r = 0.45, p < 0.001), folate (r = 0.26, p = 0.005), the n-6/n-3 fatty acid ratio (r = 0.36, p < 0.001) and iodine (r = 0.42, p < 0.001). CONCLUSION: The biochemical indicators examined in this study confirmed differences in self-reported micronutrient intake between supplement and non-supplement users for vitamin D, beta-carotene, folate, n-3 fatty acids, flavonoids and iodine.

Bone marrow-derived and peritoneal macrophages have different inflammatory response to oxLDL and M1/M2 marker expression - implications for atherosclerosis research.

Macrophages are heterogeneous and can polarize into specific subsets, e.g. pro-inflammatory M1-like and re-modelling M2-like macrophages. To determine if peritoneal macrophages (PEMs) or bone marrow derived macrophages (BMDMs) resembled aortic macrophages from ApoE-/- mice, their M1/M2 phenotype, inflammatory status, and lipid metabolism signatures were compared. oxLDL accumulation was similar in PEMs and BMDMs. On protein expression level, BMDMs showed an M2-like CD206highCD11clow profile, while cholesterol loading led to enhanced CD11c expression and reduced MCP-1 secretion. In contrast, PEMs expressed low levels of CD206 and CD11c, and responded to cholesterol loading by increasing CD11c expression and MCP-1 secretion. mRNA expression of M1/M2 markers was higher in PEMS than BMDMs, while lipid metabolism genes were similarly expressed. Whole aorta flow cytometry showed an accumulation of M2-like CD206highCD11clow macrophages in advanced versus early atherosclerotic disease in ApoE-/- mice. In isolated lesions, mRNA levels of the M2 markers Socs2, CD206, Retnla, and IL4 were downregulated with increasing disease severity. Likewise, mRNA expression of lipid metabolism genes (SREBP2, ACSL1, SRB1, DGAT1, and cpt1a) was decreased in advanced versus early lesions. In conclusion, PEMs and BMDMs are phenotypically distinct and differ from macrophages in lesions with respect to expression of M1/M2 markers and lipid metabolism genes.

Liraglutide Reduces Both Atherosclerosis and Kidney Inflammation in Moderately Uremic LDLr-/- Mice.

Chronic kidney disease (CKD) leads to uremia. CKD is characterized by a gradual increase in kidney fibrosis and loss of kidney function, which is associated with a progressive increase in risk of atherosclerosis and cardiovascular death. To prevent progression of both kidney fibrosis and atherosclerosis in uremic settings, insight into new treatment options with effects on both parameters is warranted. The GLP-1 analogue liraglutide improves glucose homeostasis, and is approved for treatment of type 2 diabetes. Animal studies suggest that GLP-1 also dampens inflammation and atherosclerosis. Our aim was to examine effects of liraglutide on kidney fibrosis and atherosclerosis in a mouse model of moderate uremia (5/6 nephrectomy (NX)). Uremic (n = 29) and sham-operated (n = 14) atherosclerosis-prone low density lipoprotein receptor knockout mice were treated with liraglutide (1000 µg/kg, s.c. once daily) or vehicle for 13 weeks. As expected, uremia increased aortic atherosclerosis. In the remnant kidneys from NX mice, flow cytometry revealed an increase in the number of monocyte-like cells (CD68+F4/80-), CD4+, and CD8+ T-cells, suggesting that moderate uremia induced kidney inflammation. Furthermore, markers of fibrosis (i.e. Col1a1 and Col3a1) were upregulated, and histological examinations showed increased glomerular diameter in NX mice. Importantly, liraglutide treatment attenuated atherosclerosis (~40%, p < 0.05) and reduced kidney inflammation in NX mice. There was no effect of liraglutide on expression of fibrosis markers and/or kidney histology. This study suggests that liraglutide has beneficial effects in a mouse model of moderate uremia by reducing atherosclerosis and attenuating kidney inflammation.

The 6-a-day study: effects of fruit and vegetables on markers of oxidative stress and antioxidative defense in healthy nonsmokers.

BACKGROUND: Fruit and vegetables contain both nutritive and nonnutritive factors that might contribute to redox (antioxidant and prooxidant) actions. OBJECTIVE: We investigated the relative influence of nutritive and nonnutritive factors in fruit and vegetables on oxidative damage and enzymatic defense. DESIGN: A 25-d intervention study with complete control of dietary intake was performed in 43 healthy male and female nonsmokers who were randomly assigned to 1 of 3 groups. In addition to a basic diet devoid of fruit and vegetables, the fruit and vegetables (Fruveg) group received 600 g fruit and vegetables/d; the placebo group received a placebo pill, and the supplement group received a vitamin pill designed to contain vitamins and minerals corresponding to those in 600 g fruit and vegetables. Biomarkers of oxidative damage to protein and lipids and of antioxidant nutrients and defense enzymes were determined before and during intervention. RESULTS: Plasma lipid oxidation lag times increased during intervention in the Fruveg and supplement groups, and the increase was significantly higher in the former. Plasma protein carbonyl formation at lysine residues also increased in both of these groups. Glutathione peroxidase activity increased in the Fruveg group only. Other markers of oxidative damage, oxidative capacity, or antioxidant defense were largely unaffected by the intervention. CONCLUSIONS: Fruit and vegetables increase erythrocyte glutathione peroxidase activity and resistance of plasma lipoproteins to oxidation more efficiently than do the vitamins and minerals that fruit and vegetables are known to contain. Plasma protein carbonyl formation at lysine residues increases because of the vitamins and minerals in fruit and vegetables.

Absorption and excretion of black currant anthocyanins in humans and watanabe heritable hyperlipidemic rabbits.

Anthocyanins are thought to protect against cardiovascular diseases. Watanabe heritable hyperlipidemic (WHHL) rabbits are hypercholesterolemic and used as a model of the development of atherosclerosis. To compare the uptake and excretion of anthocyanins in humans and WHHL rabbits, single-dose black currant anthocyanin studies were performed. Procedures for workup and analyses of urine and plasma samples containing anthocyanins were developed with high recoveries (99 and 81%, respectively) and low limits of quantification (> or =6.6 and > or =1.1 nM, respectively). The excretion and absorption of anthocyanins from black currant juice were found to be within the same order of magnitude in the two species regarding urinary excretion within the first 4 h (rabbits, 0.035%; humans, 0.072%) and t(max) (rabbits, approximately 30 min; humans, approximately 45 min). A food matrix effect was detected in rabbits, resulting in the absorption of a higher proportion of the anthocyanins from black currant juice than from an aqueous citric acid matrix. In humans the absorption and urinary excretion of anthocyanins from black currant juice were found to be proportional with dose and not influenced by the ingestion of a rice cake. In both species a larger proportion of the anthocyanin rutinosides than of the glucosides was absorbed, whereas the structure of the aglycon had no influence on the absorption and excretion. The anthocyanins had no effect in rabbits on the antioxidant capacity of plasma measured as Trolox equivalent antioxidant capacity and ferruc reducing ability of plasma.

Quantification of anthocyanins in commercial black currant juices by simple high-performance liquid chromatography. Investigation of their pH stability and antioxidative potency.

Quantitative determinations of the four black currant anthocyanins, cyanidin 3-O-beta-glucoside, cyanidin 3-O-beta-rutinoside, delphinidin 3-O-beta-glucoside, and delphinidin 3-O-beta-rutinoside, were achieved in black currant juices by a rapid and sensitive high-performance liquid chromatographic (HPLC) method. The method was validated, and quantification of anthocyanins in 13 commercially available black currant beverages was demonstrated. To optimize the handling of anthocyanin-containing samples, the pH-dependent stability of the anthocyanins was investigated. Four anthocyanins were incubated for 24 h in aqueous solutions at 13 different pH levels between 0.6 and 5.2, after which the samples were analyzed by HPLC. More than 90% of each anthocyanin remained intact up to pH 3.3. At pH 3.8 a local minimum in stability was detected, and at pH >4.5 the stability rapidly decreased. The antioxidant capacity of all 13 black currant juices was investigated by TEAC and FRAP, and the antioxidant potential of both the anthocyanin and the vitamin C contents in the juices was evaluated. This indicated that <70% of the antioxidant capacity of the juices could be attributed to the anthocyanin content or to vitamin C, signifying that other very potent antioxidants are present in commercial black currant juices.

Effect of diets based on foods from conventional versus organic production on intake and excretion of flavonoids and markers of antioxidative defense in humans.

Different food production methods may result in differences in the content of secondary metabolites such as polyphenolic compounds. The present study compared conventionally (CPD) and organically produced (OPD) diets in a human crossover intervention study (n = 16) with respect to the intake and excretion of five selected flavonoids and effect on markers of oxidative defense. The urinary excretion of quercetin and kaempferol was higher after 22 days of intake of the OPD when compared to the CPD (P < 0.05). The excretions of flavonoids in urine as a percentage of intake (0.6-4%) were similar after both interventions. Most markers of antioxidative defense did not differ between the diets, but intake of OPD resulted in an increased protein oxidation and a decreased total plasma antioxidant capacity compared to baseline (P < 0.05). Some varietal difference was seen in the study, and because selection of more resistant varieties is of central importance to organic farming, it cannot be excluded that the observed effects originate from these differences. The food production method affected the content of the major flavonoid, quercetin, in foods and also affected urinary flavonoids and markers of oxidation in humans.

Urinary flavonoid excretion and risk of acute coronary syndrome in a nested case-control study.

BACKGROUND: Epidemiologic studies have suggested that a higher intake of flavonoids may be associated with lower risk of ischemic heart disease. However, the traditional estimation of flavonoid intake by using dietary assessment methods is affected by subjective measures. OBJECTIVE: We examined whether the objective measurement of dietary flavonoids excreted in urine is associated with lower risk of acute coronary syndrome (ACS). DESIGN: A case-control study was nested in the Danish Diet, Cancer and Health cohort study. Cases were identified in participants who had received a first-time ACS diagnosis in the Danish National Patient Registry after the time of enrollment into the Diet, Cancer and Health study. The excretion of 10 flavonoids, which represent 5 subclasses, was measured in spot urine samples by using liquid chromatography-mass spectrometry. RESULTS: A total of 393 eligible cases with ACS were identified and matched to 393 noncases by using incidence density sampling. For kaempferol, most of the individual ORs were statistically significant and from 42% to 61% lower when the higher 4 quintiles were compared with the lowest quintile. The P-trend was not significant. For daidzein, individual ORs were 5-38% lower. None of the individual ORs were significant, but the P-trend was 0.041. For the remaining flavonoids, there were no significant relations between urinary excretion and risk of ACS. CONCLUSIONS: Except for kaempferol and daidzein, there were no significant associations between the urinary excretion of flavonoids and risk of ACS. A lack of relations may be a result of the use of short-term exposure measures.

Urine flavonoids and plasma carotenoids in the validation of fruit, vegetable and tea intake during pregnancy in the Norwegian Mother and Child Cohort Study (MoBa).

OBJECTIVE: To validate a new food-frequency questionnaire (FFQ) for measuring the intake of fruit, vegetables and tea reported by women participating in the Norwegian Mother and Child Cohort Study (MoBa). DESIGN: Intake of fruits, vegetables and tea estimated by the FFQ was compared with urinary flavonoid excretion, plasma carotenoid concentration and intake measured by a 4-day weighed food diary (FD). The triangular method was applied to calculate FFQ validity coefficients using two independent biomarkers. SETTING AND SUBJECTS: One hundred and nineteen women participating in MoBa. RESULTS: The FFQ estimate of fruit intake was significantly correlated with urine phloretin (r = 0.33), citrus fruit/juice with urine hesperetin (r = 0.44), cooked vegetables with plasma alpha-carotene (r = 0.37), and tea with urine kaempferol (r = 0.41) (P < 0.01 for all). On average, 60% of the participants fell into the same or adjacent quintiles when classified by FFQ and biomarkers. Significant correlations between the FFQ and FD were found for fruit (r = 0.39), vegetables (r = 0.34), juices (r = 0.50) and tea (r = 0.53). The FFQ validity coefficient was 0.65 for citrus fruit/juice and 0.59 for cooked vegetables as calculated by the triangular method. CONCLUSIONS: The validation study shows that the MoBa FFQ can be used to estimate fruit, juice, vegetable and tea intake in pregnant Norwegian women, and to rank individuals within the distribution.

Urinary excretion of total isothiocyanates from cruciferous vegetables shows high dose-response relationship and may be a useful biomarker for isothiocyanate exposure.

BACKGROUND: Isothiocyanates (ITCs), hydrolysis products from glucosinolates, are a family of biologically active compounds originating from cruciferous vegetables. Many ITCs are assumed to have cancer preventive effects and to further evaluate these potential health effects, reliable biomarkers of ITC exposure are needed. AIM OF THE STUDY: In this study we investigated the ability of urinary ITC excretion to reflect a low or high daily intake of cruciferous vegetables. METHODS: The design was a controlled human crossover study (n = 6). Subjects consumed a self-restricted glucosinolate-free diet 48 h before the study-day where a basic diet supplemented with 80 or 350 g of mixed cruciferous vegetables was consumed. All urine was collected in intervals during the 48 h period after ingestion of the cruciferous vegetables. Total ITC in the cruciferous mixture and total ITC and their metabolites in urine was quantified as the cyclocondensation product of 1,2-bezenedithiol by high performance liquid chromatography. RESULTS: The total urinary excretion of ITCs correlated significantly with the two doses of ITC from diets with high or low cruciferous content (r (s )= 0.90, P < 0.01). The fraction of urinary ITC excreted was 69.02 +/- 11.57% and 74.53 +/- 8.39% of the amounts ingested for 80 and 350 g cruciferous vegetables, respectively. CONCLUSION: The results in this study indicate that the urinary excretion of ITCs, measured by use of the cyclocondesation reaction, is a useful and precise tool that may be used as a biomarker of ITC exposure in population based studies.

Relative validity of fruit and vegetable intake estimated by the food frequency questionnaire used in the Danish National Birth Cohort.

OBJECTIVE: To validate the fruit and vegetable intake estimated from the Food Frequency Questionnaire (FFQ) used in the Danish National Birth Cohort (DNBC). SUBJECTS AND SETTING: The DNBC is a cohort of 101,042 pregnant women in Denmark, who received a FFQ by mail in gestation week 25. A validation study with 88 participants was made. A seven-day weighed food diary (FD) and three different biomarkers were employed as comparison methods. RESULTS: Significant correlations between FFQ and FD-based estimates were found for fruit (r = 0.66); vegetables (r = 0.32); juice (r = 0.52); fruit and vegetables (F&V) (r = 0.57); and fruit, vegetables, and juice (F&V&J) (r = 0.62). Sensitivities of correct classification by FFQ into the two lowest and the two highest quintiles of F&V&J intake were 58-67% and 50-74%, respectively, and specificities were 71-79% and 65-83%, respectively. F&V&J intake estimated from the FFQ correlated significantly with urinary flavonoids (r = 0.39, p<0.005), plasma beta-carotene (r = 0.21, p<0.05) and erythrocyte folate (r = 0.33, p<0.005); all average biomarker levels increased across increasing quintiles of F&V&J intake. CONCLUSION: Data on F&V&J recorded by FFQ in DNBC can be used in etiological analyses of associations between fruit and vegetable intake and health outcomes, provided that the potential misclassification of dietary exposure is kept in mind.

Dietary supplementation with an extract of lycopene-rich tomatoes does not reduce atherosclerosis in Watanabe Heritable Hyperlipidemic rabbits.

Tomatoes are rich in lycopene and other carotenoids which have shown beneficial effects on CVD in epidemiological and intervention studies. In the present study the effect of an extract of lycopene-rich tomatoes, Lyc-O-Mato on atherosclerosis was studies in Watanabe Heritable Hyperlipidemic rabbits. The rabbits were fed a control diet, a control diet supplemented with the tomato extract or a control diet supplemented with a mixture of plant oils for 16 weeks. Lycopene was detected only in plasma of rabbits receiving tomato extract. The tomato extract had no effect on cholesterol and triacylglycerol levels measured in total plasma, lipoprotein fractions and on aortic atherosclerosis evaluated biochemically and by microscopy. Oxidation of lipids in unfractionated plasma also was unaffected by the intake of tomato extract. In conclusion, the tomato extract increased plasma levels of lycopene in rabbits, but had no effect on hypercholesterolaemia, oxidation of plasma lipids or aortic atherosclerosis.

Bioavailability is improved by enzymatic modification of the citrus flavonoid hesperidin in humans: a randomized, double-blind, crossover trial.

Hesperidin is the predominant polyphenol consumed from citrus fruits and juices. However, hesperidin is proposed to have limited bioavailability due to the rutinoside moiety attached to the flavonoid. The aim of this study was to demonstrate in human subjects that the removal of the rhamnose group to yield the corresponding flavonoid glucoside (i.e., hesperetin-7-glucoside) will improve the bioavailability of the aglycone hesperetin. Healthy volunteers (n=16) completed the double-blind, randomized, crossover study. Subjects randomly consumed hesperetin equivalents supplied as orange juice with natural hesperidin ("low dose"), orange juice treated with hesperidinase enzyme to yield hesperetin-7-glucoside, and orange juice fortified to obtain 3 times more hesperidin than naturally present ("high dose"). The area under the curve (AUC) for total plasma hesperetin of subjects consuming hesperetin-7-glucoside juice was 2-fold higher than that of subjects consuming the "low" dose hesperidin juice [3.45+/-1.27 vs. 1.16+/-0.52 mmol/(L.h), respectively, P>0.0001]. The AUC for hesperetin after consuming the hesperetin-7-glucoside juice was improved to the level of the "high" dose hesperidin juice [4.16+/-1.50 mmol/(L.h)]. The peak plasma concentrations (C(max)) of hesperetin were 4-fold higher (2.60+/-1.07 mmol/L, P<0.0001) after subjects consumed hesperetin-7-glucoside juice compared with those consuming "low" dose hesperidin juice (0.48 +/- 0.27 mmol/L), and 1.5-fold higher than those consuming "high" dose hesperidin juice (1.05+/-0.25 mmol/L). The corresponding T(max) was much faster (0.6+/-0.1 h, P<0.0001) after subjects consumed hesperetin-7-glucoside juice compared with "low" dose (7.0+/-3.0 h) and "high" dose (7.4+/-2.0 h) hesperidin juices. The results of this study demonstrated that the bioavailability of hesperidin was modulated by enzymatic conversion to hesperetin-7-glucoside, thus changing the absorption site from the colon to the small intestine. This may affect future interventions concerning the health benefits of citrus flavonoids.

Biological effects of fruit and vegetables.

A strong and persistent effect of plant-derived foods on the prevention of lifestyle diseases has emerged from observational studies. Several groups of constituents in plants have been identified as potentially health promoting in animal studies, including cholesterol-lowering factors, antioxidants, enzyme inducers, apoptosis inducers etc. In human intervention studies the dose levels achieved tend to be lower than the levels found to be effective in animals and sampling from target organs is often not possible. A controlled dietary human intervention study was performed with forty-three volunteers, providing 600 g fruit and vegetables/d or in the controls a carbohydrate-rich drink to balance energy intake. Surrogate markers of oxidative damage to DNA, protein and lipids, enzymic defence and lipid metabolism were determined in blood and urine. It was found that a high intake of fruit and vegetables tends to increase the stability of lipids towards oxidative damage. Markers of oxidative enzymes indicate a steady increase in glutathione peroxidase (GPX1) activity in erythrocytes during intervention with fruit and vegetables but there is no effect on GPX1 transcription levels in leucocytes. No change occurs in glutathione-conjugating or -reducing enzyme activities in erythrocytes or plasma, and there are no effects on the transcription of genes involved in phase 2 enzyme induction or DNA repair in leucocytes. Fruit and vegetable intake decreases the level of total cholesterol and LDL-cholesterol, but does not affect sex hormones. In conclusion, it has been shown that total cholesterol and LDL-cholesterol, markers of peripheral lipid oxidation, and erythrocyte GPX1 activity are affected by high intakes of fruit and vegetables. This finding provides support for a protective role of dietary fruit and vegetables against CVD.

Urinary total flavonoid excretion but not 4-pyridoxic acid or potassium can be used as a biomarker for the intake of fruits and vegetables.

To gain better insight into the potential health effects of fruits and vegetables, reliable biomarkers of intake are needed. The main purpose of this study was to investigate the ability of flavonoid excretion in both 24-h and morning urine samples to reflect a low intake and moderate changes in fruit and vegetable consumption. Furthermore, the urinary excretions of 4-pyridoxic acid (4-PA) and potassium were investigated as other potential biomarkers of fruit and vegetable intake. The study was designed as a 5-d randomized, controlled crossover study. On d 1-3, the men (n = 12) consumed a self-restricted flavonoid-free diet. On d 4, they were provided a strictly controlled diet containing no fruits or vegetables (basic diet). On d 5, they consumed the basic diet supplemented with 300 or 600 g of fruits and vegetables. The total excretion of flavonoids in 24-h urine samples increased linearly with increasing fruit and vegetable intakes (r(s) = 0.86, P < 1 x 10(-6)). The total excretion of flavonoids in morning urine also increased, but the association was weaker (r(s) = 0.59, P < 0.0001). Urinary 4-PA in 24-h and morning urine samples increased significantly only with the 600-g increase in fruit and vegetable intake, whereas the excretion of potassium in urine did not reflect the changes in fruit and vegetable intake. We conclude that the total excretion of flavonoids in 24-h urine may be used as a new biomarker for fruit and vegetable intake.

In vitro metabolism of genistein and tangeretin by human and murine cytochrome P450s.

Recombinant cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6 enzymes obtained from Escherichia coli and human liver microsomes samples were used to investigate the ability of human CYP enzymes to metabolize the two dietary flavonoids, genistein and tangeretin. Analysis of the metabolic profile from incubations with genistein and human liver microsomes revealed the production of five different metabolites, of which three were obtained in sufficient amounts to allow a more detailed elucidation of the structure. One of these metabolites was identified as orobol, the 3'-hydroxylated metabolite of genistein. The remaining two metabolites were also hydroxylated metabolites as evidenced by LC/MS. Orobol was the only metabolite formed after incubation with CYP1A2. The two major product peaks after incubation of tangeretin with human microsomes were identical with 4'-hydroxy-5,6,7,8-tetramethoxyflavone and 5,6-dihydroxy-4',7,8-trimethoxyflavone, previously identified in rat urine in our laboratory. By comparison with UV spectra and LC/MS fragmentation patterns of previously obtained standards, the remaining metabolites eluting after 14, 17 and 20 min. were found to be demethylated at the 4',7-, 4',6-positions or hydroxylated at the 3'- and demethylated at the 4'-positions, respectively. Metabolism of tangeretin by recombinant CYP1A2, 3A4, 2D6 and 2C9 resulted in metabolic profiles that qualitatively were identical to those observed in the human microsomes. Inclusion of the CYP1A2 inhibitor fluvoxamine in the incubation mixture with human liver microsomes resulted in potent inhibition of tangeretin and genistein metabolism. Other isozymes-selective CYP inhibitors had only minor effects on tangeretin or genistein metabolism. Overall the presented observations suggest major involvement of CYP1A2 in the hepatic metabolism of these two flavonoids.