Urinary Excretion of Flavonoids Reflects Even Small Changes in the Dietary Intake of Fruits and Vegetables

Cancer Epidemiology, Biomarkers & Prevention 843

Asgeir Brevik,1 Salka Elbøl Rasmussen,2 Christian A. Drevon,1 and Lene Frost Andersen1 1Institute for Nutrition Research, School of Medicine, University of Oslo, Oslo, Norway and 2Department of Toxicology and Risk Assessment, Danish Institute for Food and Veterinary Research, Søborg, Denmark

Abstract

Background: Due to the random and systematic mea- Following a 2-week washout and a 1 week run-in pe- surement errors associated with current dietary assess- riod, the regimens were switched between the groups. ment instruments, there is a need to develop more Results: An increased intake of mixed fruits and vegeta- objective methods of measuring the intake of foods of bles from 2 to 5 servings per day significantly enhanced importance to human health. Objective: The purpose urinary excretion of eriodictyol, naringenin, hesperetin, of this study was to test whether urinary excretion of quercetin, kaempferol, isorhamnetin, and tamarixetin. flavonoids could be used to identify subjects who are The citrus flavonoids naringenin and hesperetin meeting Norwegian recommendations for fruit and showed a steep dose-response relationship to dietary vegetable intake (5 servings per day) from individuals intake of fruits and vegetables, whereas the associa- who are consuming the national average amount of tion to eriodictyol, quercetin, kaempferol, isorhamne- fruits and vegetables (2 servings per day). Design: tin, and tamarixetin was more moderate. Conclusion: Twenty-four-hour urine samples were collected in a The present study indicates that urinary excretion of strict crossover controlled feeding study. Forty healthy dietary flavonoids may be used to assess changes of subjects (19–34 years) were included in the study. After mixed fruit and vegetable intake corresponding to an a 1-week run-in period, one group was given a con- increase from the present national intake in Norway trolled diet that included 2 servings (300 g) of fruits to the recommended amount of 5 servings of fruits and vegetables daily for 14 days, while the other group and vegetables daily. (Cancer Epidemiol Biomarkers was given a diet containing 5 servings (750 g) per day. Prev 2004;13(5):843–9)

Introduction

High intake of fruits and vegetables has been shown to cultural and geographic variation in eating patterns, protect against development of many non-communica- the validation of biomarkers for food items will have to ble diseases like several types of cancers and coronary include several controlled studies testing a large variety heart disease (1–4). Accurate estimation of fruit and of eating patterns. vegetable intake is critical to further study the associ- Several substances found in fruits and vegetables may ation between intake and development of chronic be potential biomarkers, and serum concentrations of disease. Food diaries, food frequency questionnaires, carotenoids and vitamin C have received most attention and dietary recalls represent traditional methods for (10–18). However, these biomarkers have several limi- dietary assessment. All of these methods are associated tations; the absorption of carotenoids is subject to high with large random and systematic errors (5–9). Blood inter-individual variation (19, 20), and is affected by and urine biomarkers for intake of foods may offer a factors such as gender, body mass index, physical ac- more objective, universal, and physiologically relevant tivity, and amount of fat in the diet (21–25). Moreover, method for measuring intake. However, a thorough plasma concentration of vitamin C has been shown to validation of the suggested biomarkers represents a be affected by smoking and oxidative status (26). New critical and often underrated step in the development candidate biomarkers are the flavonoids that are found and maturation of new biomarkers. Due to large ubiquitously in most fruits and vegetables. A recent parallel feeding study has demonstrated a correlation between total urinary excretion of flavonoids and the Received 7/11/03; revised 1/6/04; accepted 1/16/04. intake of fruits, berries, and vegetables (27). Beyond this Grant support: Norwegian Research Council, National Nutrition Council, Throne study, the literature about flavonoids as biomarker of Holst Foundation for Nutrition Research, and Freia Chokoladefabriks fruit and vegetable intake is scarce. Medisinske Fond. The costs of publication of this article were defrayed in part by the payment of The aim of the present study was to investigate page charges. This article must therefore be hereby marked advertisement in whether urinary excretion of flavonoids could be used accordance with 18 U.S.C. Section 1734 solely to indicate this fact. as biomarkers for changes in intake of fruits and Requests for reprints: Asgeir Brevik, Institute for Nutrition Research, School of Medicine, University of Oslo, P. O. Box 1046, 0316 Oslo, Norway. vegetables from the Norwegian average consumption Phone: 47-22-85-15-27; Fax: 47-22-85-13-98. E-mail: [email protected] (2 servings per day) to the recommended consumption

Cancer Epidemiol Biomarkers Prev 2004;13(5). May 2004

of fruits and vegetables (5 servings per day). A consider- period, lasting 14 days, while group B’s diet in this able inter-individual variation in flavonoid uptake and period contained 5 servings of fruits and vegetables metabolism has been reported (28–30), and to reduce the (750 g). Group A and B switched to 5 and 2 servings of importance of this variation, a crossover design was used. fruits and vegetables, respectively, in the second 14-day diet period. The two diet periods were separated by a washout period of 14 days. During washout, the Materials and Methods participants consumed a normal diet. Before each diet period, all participants had to go through a 7-day run- The dietary intervention was carried out at the Institute in period on a self-selected diet without fruits and veg- for Nutrition Research at the School of Medicine, etables. The run-in periods were included as an attempt University of Oslo. The regional branch of The National to bring all participants to similar level with respect to Committee for Medical Research Ethics approved the baseline intake of fruits and vegetables. Thus, the overall study protocol. study design was 1 week (run-in) + 2 weeks (diet period Subjects were recruited among students from the 1) + 2 weeks (washout) + 1 week (run-in) + 2 weeks (diet Medical faculty, University of Oslo, Norway. Inclusion period 2). criteria were a body mass index <30 kg/m2, alcohol con- Diet Composition. All subjects had diets with energy sumption <30 g/day, no use of vitamins or other food levels related to their energy needs. The relative content supplements the previous 2 months, age <35 years, no of energy from protein, fat, and carbohydrates was allergies, no prescribed medication (except contracep- similar for all energy levels. The energy intake from tives), no smoking, and no major fluctuations in body breakfast, lunch, and snack meals was used to adjust the weight (<2 kg) during the last 2 years before baseline. energy intake. Except from a small amount of jam, these Body mass index was calculated as weight (kilograms) meals were free of fruits and vegetables. Dinner and all divided by the height squared (meters). One person the included fruits and vegetables were served and eaten dropped out during the washout period between the two under supervision of researchers at the Institute for diet periods, and the total number of participants Nutrition Research. Participants were free to choose completing both dietary periods was 39. A crossover where they wanted to consume their breakfast, lunch, analysis is based on within-subject differences, the and snack meals. rationale being that subjects serve as their own control, For breakfast and lunch, subjects were given bread or and the analysis requires whole data series. Furthermore, cereals along with small servings of jam and margarine. one outlier was discarded from the flavonoid analysis To escort the bread, different sorts of cheese and ham because of extremely high baseline values. Data from 38 could be selected from a few predefined alternatives. subjects were included in data evaluation. Baseline Cake, buns, and chocolate (Saturday) were provided as characteristics of the participants are shown in Table 1. snacks. Dinners were cycled between the following six Study Protocol. We performed a randomized cross- different dishes: pasta carbonara, hash, baked fish, meat over study in the fall of 2001. Participants were stratified and potato, smoked salmon with sauce, and chicken on the basis of gender and randomized into groups A curry with rice. All food items eaten in the two diet pe- and B. Group A was assigned to a diet containing 2 riods were supplied except for milk and spreads, which servings of fruits and vegetables (300 g) in the first diet the participants were allowed to choose every day from

Table 1. Baseline characteristics of participants in dietary intervention with fruits and vegetables

Group A* Group Bc Pooled baseline

N 18 20 38 Males 8 10 18 Females 10 10 20 Age (years), x¯ (range) 24 (20.0–34.0) 22.5 (19.0–30.0) 23.2 (19.0–34.0) Body mass index (kg/m2), x¯ (range)b 21.8 (19.3–26.3) 22.5 (18.3–27.8) 22.2 (18.3–27.8) Basal metabolic rate (MJ), x¯ (range)x 6.5 (5.2–8.8) 6.8 (5.0–8.6) 6.7 (5.0–8.8)

Mean (SD)

Phloretin (ng/ml) 47.9 (48.1) 33.0 (42.7) 40.5 (34.5) Eriodictyol (ng/ml) 15.4 (26.2) 17.4 (24.0) 16.4 (20.8) Naringenin (ng/ml) 32.0 (29.0) 22.3 (18.4) 27.1 (17.0) Hesperetin (ng/ml) 9.2 (9.0) 9.7 (9.1) 9.4 (6.6) Quercetin (ng/ml) 11.6 (10.9) 11.2 (11.0) 11.4 (7.7) Kaempferol (ng/ml) 7.7 (9.3) 8.8 (12.0) 8.3 (7.5) Isorhamnetin (ng/ml) 4.9 (1.0) 4.9 (1.7) 4.9 (1.0) Tamariexetin (ng/ml) 5.0 (1.3) 4.9 (2.4) 5.0 (1.3)

Note: Flavonoids were measured in 24-h urine samples. *Group A was given 2 servings of fruits and vegetables in the first period and 5 servings in the second period. c Group B was given 5 servings in the first period and 2 servings in the second period. b BMI was calculated as weight (kilograms) divided by the height squared (meters). xBMR was calculated using WHO standard formulas (38).

Cancer Epidemiol Biomarkers Prev 2004;13(5). May 2004

a few alternatives. No additional foods or beverages, of each of the two diet periods. All subjects collected a except for water, coffee, and tea, were allowed. Tea bags total of four 24-h urine samples. Aliquots of 10 ml (10%) (one brand) were given to the participants, and they had aqueous ascorbic acid and 50 ml (1 M) HCl were pre- to record the number of tea bags used and the amount of weighed in each sample container (2.5 l). In addition to tea consumed during the diet periods. On weekends, all the main sample container, all subjects were given a foods were supplied frozen in insulated plastic thermo- smaller 500 ml container with 2 ml (10%) aqueous bags, except from a plate of milk chocolate which was ascorbic and 10 ml (1 M) HCl that was easier to carry handed out along with the weekend food packages. around. Total urine volumes were measured on delivery The types of fruits and vegetables included are listed of the urine samples, and six 1-ml samples were stored inTable2.Theywerechosentomatchatypical at 70jC until analysis. Norwegian diet (31, 32). All fruits and vegetables were Reagents and Standards. Acetonitrile and methanol from the same batch, and all fruit and vegetable servings were of HPLC grade and obtained from Rathburne Ltd. were pre-prepared before the intervention and frozen (Walkerburn, United Kingdom). The flavonoid standards at 20jC. The vegetables were lightly heated before kaempferol, isorhamnetin, tamarixetin, genistein, and serving. Nutrient losses during the freezing period were daidzein were all obtained from Apin Chemicals Ltd. not measured. The relative amounts of different fruits (Oxon, United Kingdom). Phloretin and hesperetin were and vegetables were the same in both the high and low purchased from Sigma Chemicals Co. (St. Louis, MO) fruitandvegetabledietperiod,andthefruitand and quercetin and naringenin were from Aldrich vegetable combination served daily during a diet period (Steinheim, Germany). The isotopic-labelled internal was the same. Only the non-fruit and vegetable com- standards, 3 13C labelled genistein and 3 13C labelled ponent of the dinner were cycled between six alter- daidzein, were obtained from School of Chemistry, natives. All participants had the same main course for University of St. Andrews, United Kingdom. All stand- dinner on the same day. At the end of each diet period, ards were HPLC grade. A stock solution of 100 Ag/ml of all subjects had to report in a written diary if they had a mixture of all the flavonoid standards was prepared in eaten all the supplied foods, what they had not eaten, DMSO. Stock solutions of the internal standards genis- and if they had eaten additional food not allowed tein, 3 13C labelled genistein, daidzein, and 3 13C according to the protocol. The written diaries also con- labelled daidzein were prepared in DMSO, at concen- tained information about ad libitum consumption of tea trations of 20 Ag/ml. All stock solutions were stored and coffee during the diet periods. at 20jC and were stable for at least 3 months. The Energy requirements were calculated on the basis of enzymes used for enzymatic hydrolysis of the urine basal metabolic rate and patterns of physical activity samples were h-glucuronidase (Escherichia coli,>200 obtained from a personal interview (33). Basal metabolic standard units/ml) obtained from Boehringer Mannheim rate was calculated using standard formulas (34). During (Mannheim, Germany) and arylsulfatase (Aerobacter the diet periods, all subjects were weighed 3 times aerogenes, 16.8 standard units/ml) from Sigma (St. Louis, weekly. Dietary intake of total energy from fat, protein, MO). All other chemicals used were of HPLC or reagent and carbohydrates in the diet were calculated using a grade. newly developed software tool (KBS, version 3.1, 2002) linked to the national food composition table. All Determination of Flavonoids. The urinary concen- unconsumed food items were subtracted and data from trations of eight dietary flavonoids were determined by the participant’s actual consumption were used for HPLC-mass spectrometry (MS) using atmospheric pres- calculation of total energy intake. sure chemical ionization (APCI). The flavanones: eriodictyol, naringenin, and hesperetin; the flavonols: quercetin, Collection of Urine Samples. Urine samples were kaempferol, isorhamnetin, and tamarixetin; and the di- collected on the day before the start and on the last day hydrochalcone phloretin were all determined simulta- neously in each urine sample. The method is essentially Table 2. Fruits and vegetables included in the described elsewhere (28), except for the inclusion of experimental diet the determination of eriodictyol and the use of different internal standards in the present study. Eriodictyol was Foods Low FV Diet High FV Diet eluted with a retention time of 15.2 min and was detected (g/day) (g/day) at m/z 287 [M1], and the fragment ion at m/z 151 Raspberry 6.0 15.0 was used as qualifier ion. In brief, 2-ml aliquots of the Blueberry 6.0 15.0 24-h urine samples from each individual were added to Red pepper (cooked) 8.9 22.5 25 Al of a mixture of 500 ng of genistein and 500 ng Pear 10.8 27.0 3 13C labelled genistein dissolved in DMSO as internal Canned tomatoes (cooked) 11.0 27.7 standards and enzymatically hydrolyzed as described Jam (strawberry) 12.0 30.0 elsewhere (28) Genistein and 3 13C labelled genistein Apple 19.4 48.6 Orange 20.5 51.3 was eluted at 22.6 min and was determined as [M1] , Onion (cooked) 20.7 51.9 at m/z 269 and 272, respectively. After hydrolysis, 2 ml Tomato (cooked) 26.9 67.4 ice-cold methanol were added to each sample to stop the Broccoli (cooked) 27.5 90.0 reaction, and the samples were evaporated to dryness Cauliflower (cooked) 27.5 47.0 under vacuum. The hydrolyzed samples were redis- Orange juice 46.4 116.1 Carrot (cooked) 56.0 140.0 solved in 10% aqueous methanol, 1% formic acid, and Total fruits and vegetables 300.0 750.0 added 25 Al of a mixture of 500 ng of daidzein and 500 ng 3 13C labelled daidzein dissolved in DMSO

Cancer Epidemiol Biomarkers Prev 2004;13(5). May 2004

as additional internal standards, assessing the perfor- for these instances, all subjects were completely compli- mance of the mass spectrometer, giving a final volume ant to all dietary restriction and to the dietary treatments. of 250 Al. Daidzein and the isotopic-labelled daidzein All subjects had constant body weight during the study were detected at a retention time of 17.9 min and as the (<1.5 kg fluctuation). molecular ion [M1] at m/z 269 and 272, respectively. Tea and coffee consumption did not differ in groups The sample was then centrifuged at 10,000 g for A and B during the diet periods, and was not related to 5 min at 4jC and the entire amount of the supernatant the urinary excretion of flavonoids. was injected onto the HPLC-APCI-MS system. Before, Consumption of the diet containing the recommended and after each series of analysis, the performance of the amount of 5 servings of fruits and vegetables per entire LC-MS assay was controlled by injections of ali- day gave significantly higher urinary excretion of the quots containing all employed flavonoid standards, in- flavanones eriodictyol, naringenin, hesperetin and the cluding the internal standards as previously described flavonols quercetin, kaempferol, isorhamnetin, and tam- (28). In addition, blank urine samples added to 250 ng ariexetin compared to the diet containing 2 servings of of each of the analytes and the four internal standards fruits and vegetables per day. Period effects were (500 ng/sample) were included in the beginning, in the observed for tamariexetin and isorhamnetin and iso- middle, and in the end of each series of 25 urine sam- rhamnetin was afflicted with a period-treatment inter- ples to evaluate performance of each run. The coef- action (Table 4). The statistical method automatically ficient of variation % (CV%) of the repeatability of the adjusts for period effects. assay was below 12% (n = 20). The laboratory person- Figure 1 shows the absolute excretion in 24 h urine of nel were blinded for all treatments. all flavonoids measured at three periods; at baseline after the first run-in period where no fruit and vegetable Statistical Analysis. Data were ln transformed to were consumed, after the diet period with 2 servings of obtain normality in the crossover analysis. The limit of fruit and vegetable per day, and after the diet period quantification for all flavonoids determined (LOQ) in with 5 servings per day. All flavonoids with a significant the LC-MS analysis was 5 ng/ml urine. treatment effect in the crossover analyses demonstrated The analyses of a two-treatment crossover study can a dose-dependent increase in urinary excretion from be presented as three two-sample t tests (35, 36). Before baseline to the highest serving size (Fig. 1). The dose- comparing the treatments, the possibility of a period response relationship was strongest for quercetin, iso- effect and a treatment interaction effect (carry-over effect) rhamnetin, naringenin, hesperetin, and total flavonoids. were tested. A potential period effect was tested by a Moreover, the urinary concentrations of flavonols were two-sample t test to compare differences between the lower than the flavanones. Naringenin and hesperetin diet periods in the two groups of participants. Possible responded more than any other flavonoid to the dietary treatment-period interaction was investigated by notic- treatment. The increase in flavonoid excretion compared ing that in the absence of an interaction, a subject’s to baseline was only significant after the highest fruit average response to the two diets should be similar and vegetable dose (5 servings) for kaempferol, tamar- regardless of the order in which the diets were given. The ixetin, and eriodictyol. Phloretin did not respond at all test for interaction is thus a two-sample t test comparing to the intervention. average for group A with average for group B. Because the two crossover groups were not the same size, the effect of the experimental diet was tested by performing Discussion a two-sample t test to compare the average differences between the two treatments. Mann-Whitney U test was The main finding in this controlled crossover study is used to test for possible differences between groups in that urinary excretion of several flavonoids may be used the non-transformed baseline values. Most commenta- to assess changes in intake of mixed fruits and vegetables tors would agree that the statistical procedure employed corresponding to an increase from 2 servings to the adjusts the treatment effect adequately for any differ- recommended amount of 5 servings of mixed fruits and ence due to periods and also eliminates the effect of in- vegetables per day. For most of the flavonoids measured, dividual subjects from the estimate of the SE of the there was also a significant increase from baseline to 2 treatment (37). The significance level was set to 5%, and servings (Fig. 1). To our knowledge, this is the first all analyses were performed using SPSS 11. time the flavanone eriodictyol has been quantified in human urine.

Results Table 3. Dietary composition during intervention

There were no significant differences between the 2 Servings/day 5 Servings/day baseline characteristics of the subjects allocated to group (n = 38) (n = 38) A or B, as seen in Table 1. The content of major nutrients Means (SD) and the energy intake was equal in the two diets providing either 2 or 5 servings of fruits and vegetables Fat, % of energy (SD) 31.9 (1.7) 32.3 (1.7) (Table 3). Compliance was good as judged from the Protein, % of energy (SD) 18.0 (0.9) 17.8 (1.0) written records and personal feedback. All experimental Carbohydrate, 50.1 (2.4) 50.0 (2.3) fruits and vegetables were consumed, except from one % of energy (SD) Energy intake, MJ (SD) 13.1 (3.1) 13.2 (3.1) portion of 123 g of fruit salad forgotten by one subject Fiber, g/day (SD) 35.4 (11.4) 43.7 (9.8) in the first diet period. Two subjects consumed a mouth- Tea, ml/day 219 (197) 177 (135) ful of communion wine twice in each diet period. Except

Cancer Epidemiol Biomarkers Prev 2004;13(5). May 2004

Table 4. Effects of crossover intervention with 2 rhamnetin (Table 4). The flavonols are found in onions, and 5 portions of fruits and vegetables on urinary apples, tea, cruciferous vegetables, and wine (40). In an excretion of flavonoids* attempt to calculate the intake of quercetin in the c Period-effect Period-treatment Treatment Netherlands (40), tea turned out to be the major source b interaction effectx (48% of total) followed by onions (29%) and apples (7%). However, no significant relationship between average Results are expressed as P values intake of tea and urinary excretion of either quercetin or kaempferol was observed in this study. This is in contrast ln Phloretin 0.807 0.994 0.082 ln Eriodictyol 0.848 0.284 0.000 to what have been reported by others (27, 40, 41). The ln Naringenin 0.208 0.989 0.000 period effect and the generally low amount of excretion ln Hesperetin 0.769 0.934 0.000 observed for tamarixetin and isorhamnetin, together ln Quercetin 0.742 0.070 0.012 with the period treatment interaction observed for ln Kaempferol 0.058 0.674 0.008 isorhamnertin, indicates that tamarixetin and isorham- ln Isorhamnetin 0.034 0.011 0.014 netin may be less stable biomarkers for mixed fruit and ln Tamariexetin 0.009 0.275 0.001 ln Total flavonoids 0.484 0.777 0.000 vegetable intake than for example quercetin. This is probably due to the fact that the urinary excretion of *Two-sample t tests are used to compare differences in the two groups of both tamarixetin and isorhamnetin not only originates participants. from the dietary intake, but are also produced as minor cGroup A (2 – 5 servings) is tested against Group B (2 – 5 servings). b endogenous metabolites of quercetin (42). Group A (Period 1 + Period 2)/2 is tested against Group B (Period 1 + There was no significant effect of serving size on uri- Period 2)/2. xGroup A (Period 1 Period 2) is tested against Group B (Period 1 nary excretion of the dihydrochalcone phloretin in this Period 2). crossover analysis. Phloretin is primarily found in apples (27, 39). Our 2 servings diet contained 19.5 g apples, whereas our 5 servings diet contained 48.5 g. Both of these There was a significant effect of number of servings servings represent a low daily intake of apples, which on the urinary excretion of the flavanones naringenin, may explain the poor response of phloretin in this study hesperetin, and eriodictyol (Table 4). The flavanones as compared to the previous study by Nielsen et al. (27). naringenin, hesperetin, and eriodictyol are abundant A reliable dose-response calculation between the fruit in citrus fruits and may thus be referred to as citrus and vegetable mixture included in the present study and flavonoids (27, 38, 39). The diet including 2 servings urinary excretion of flavonoids should certainly have of fruits and vegetables contained 67 g of orange and included more than three different dose measurements, orange juice per day, whereas the diet with 5 servings of but nevertheless, the present data seem to indicate that fruits and vegetables contained 167 g orange and orange urinary excretion of the flavanones naringenin and juice per day. Flavanone half-lives are short. In an ex- hesperetin have a steep and consistent dose-response periment performed by Erlund et al. (29), 63% of total relationship, whereas urinary excretion of the flavonols urinary excretion of naringenin and 65% of hesperetin appears to exhibit a less pronounced dose dependency were excreted in the 4–8 h fraction. (Fig. 1). Still, this more moderate response of the fla- We also observed an effect of serving size on the vonols does not necessarily mean that they are without flavonols quercetin, kaempferol, tamarixetin, and iso- interest as potential indicators of fruit and vegetable

Figure 1. Twenty-four-hour urinary excretion at baseline, 2 servings, and 5 servings, respectively. *, Differ- ent from baseline, P < 0.05. **, Different from baseline, P < 0.0001. #, Different from 2 servings, P < 0.05. ##, Different from 2 servings, P < 0.0001. Columns, medians; bars, 75% percentiles.

Cancer Epidemiol Biomarkers Prev 2004;13(5). May 2004

intake. Compared with the flavanones, the flavonols are The present study demonstrates that subjects consum- present in a wider range of fruits and vegetables, and ing the Norwegian average of 2 servings of fruits and may thus be useful as a more general marker of fruits vegetables (300 g) have a significantly lower 24 h urinary and vegetables than the former group. As judged from excretion of the flavonoids eriodictyol, naringenin, the present study, there seems to be a robust linear hesperetin, quercetin, kaempferol, and tamarixetin than relationship between the intake of the fruit and vegetable subjects consuming the recommended amount of 5 serv- mixture used in the present study and urinary excretion ings (750 g). The strongest effect was observed for nar- of quercetin. ingenin and hesperetin, whereas the weakest effect was Eriodictyol, naringenin and all flavonoids combined observed for tamarixetin. Urinary excretion of flavo- seem to demonstrate an almost linear response to serving noids is a promising biomarker for the intake of fruits size increments (Fig. 1). The urinary excretion of hes- and vegetables. peretin seems to respond more than expected in the 2–5 servings interval, whereas quercetin seems to respond stronger in the 0–2 servings interval. On the basis of Acknowledgments the present results and the results of Nielsen et al. (27), total flavonoids may seem to be a robust flavonoid We thank assistant professor Kerstin Ulla Trygg for her help with hosting our participants, serving, and preparing the food. parameter for mixed fruit and vegetable intake. How- We thank bioengineers Borghild Arntsen, Anne Randi Alvestad, ever, because the concept of ‘‘total flavonoids’’ is poorly and Hanne Schulz for collection of blood samples. defined, it remains to be agreed on what flavonoids should be included in this generic term. The strongest effect for single flavonoids in the References present study was observed for the citrus flavonoids 1. WHO Study Group on Diet, N. a. P. o. N. D. Diet, nutrition and the naringenin and hesperetin. In addition, studies have prevention of chronic diseases: executive summary. Geneva: WHO; shown a much higher excretion of ingested citrus 1991. flavonoids in the urine (up to 30%) (29) compared with 2. Steinmetz KA, Potter JD. Vegetables, fruit, and cancer. I. Epidemiol Cancer Causes Control 1991;2:325-57. other flavonoids (1–2%) (40, 43, 44). This indicates that 3. Willett WC. Diet, nutrition, and avoidable cancer. Environ Health the flavanones may be the group of flavonoids that Perspect 1995;103 Suppl 8:165-70. have the most obvious potential as nutritional bio- 4. Doll R. Chronic and degenerative disease: major causes of morbidity markers. However, the limitation is that the flavanones and death. Am J Clin Nutr 1995;62:1301S-5S. 5. Cullen KW, Baranowski T, Baranowski J, Hebert D, de Moor C. Pilot may be markers of citrus intake only and not the total study of the validity and reliability of brief fruit, juice and vegetable fruit and vegetable intake. In a nationwide study among screeners among inner city African-American boys and 17 to 20 year Norwegian adults, there are a correlation between citrus old adults. J Am Coll Nutr 1999;18:442-50. intake and total fruit and vegetable intake (r = 0.40), 6. Goldbohm RA, van’t Veer P, van den Brandt PA, et al. Reproduci- suggesting that flavanones could be used as a marker of bility of a food frequency questionnaire and stability of dietary habits 2 determined from five annually repeated measurements. Eur J Clin fruits and vegetables at least in Norway. Nutr 1995;49:420-9. The current average intake of fruits and vegetables in 7. Kipnis V, Midthune D, Freedman LS, et al. Empirical evidence of Norway is similar to the average intake in several other correlated biases in dietary assessment instruments and its implications. Am J Epidemiol 2001;153:394-403. European countries (45). However, the type of fruit and 8. Kipnis V, Schatzkin A, Midthune D, Subar A, Troiano RP. vegetable included in the diet in the present interven- Comparison of a food frequency questionnaire with 24 hour recall tion was combined to match a typical Norwegian diet, for use in epidemiologic studies: results from the observing protein which naturally may limit the generalization of the and energy nutrition (OPEN) study. The Fifth International Con- ference on Dietary Assessment Methods; 2003; Chiang Rai, Thailand. results from the study to other countries. Furthermore, 9. Kipnis V, Carroll RJ, Freedman LS, Li L. Implications of a new dietary in a free living Norwegian population, the combination measurement error model for estimation of relative risk: application of fruit and vegetable eaten would vary substantially to four calibration studies. Am J Epidemiol 1999;150:642-51. and one could easily eat ‘‘5 a day’’ on a menu exhib- 10. Block G, Norkus E, Hudes M, Mandel S, Helzlsouer K. Which plasma antioxidants are most related to fruit and vegetable consumption? iting less diversity with respect to food items than Am J Epidemiol 2001;154:1113-8. chosen for the mixed fruit and vegetable diet included 11. Campbell DR, Gross MD, Martini MC, Grandits GA, Slavin JL, Potter in the present study. JD. Plasma carotenoids as biomarkers of vegetable and fruit intake. There are several challenges associated with the use of Cancer Epidemiol Biomark Prev 1994;3:493-500. 12. McEligot AJ, Rock CL, Flatt SW, Newman V, Faerber S, Pierce JP. flavonoids as nutritional biomarkers. Uptake of flavo- Plasma carotenoids are biomarkers of long-term high vegetable noids is affected by the gut microflora, and a substantial intake in women with breast cancer. J Nutr 1999;129:2258-63. inter-individual variation in uptake has been observed 13. van Kappel AL, Steghens JP, Zeleniuch-Jacquotte A, Chajes V, (29, 30, 46). In addition to the observed variation between Toniolo P, Riboli E. Serum carotenoids as biomarkers of fruit and vegetable consumption in the New York Women’s Health Study. individuals, bioavailability of one compound may also Public Health Nutr 2001;4:829-35. vary between different foods. Hollman and Katan (41) 14. Le Marchand L, Hankin JH, Carter FS, et al. A pilot study on the use found that the bioavailability of quercetin from apples of plasma carotenoids and ascorbic acid as markers of compliance was only 30% of that from onions. Although the to a high fruit and vegetable dietary intervention. Cancer Epidemiol Biomark Prev 1994;3:245-51. flavonoid uptake differs between subjects, the flavonoid 15. Rock CL, Swendseid ME, Jacob RA, McKee RW. Plasma carotenoid biomarker reveals the true systemic exposure of individ- levels in human subjects fed a low carotenoid diet. J Nutr 1992;122: uals, which are the most central parameter in relation to 96-100. the disease protective effect of dietary flavonoids. 16. Martini MC, Campbell DR, Gross MD, Grandits GA, Potter JD, Slavin JL. Plasma carotenoids as biomarkers of vegetable intake: the University of Minnesota Cancer Prevention Research Unit Feeding Studies. Cancer Epidemiol Biomark Prev 1995;4:491-6. 17. Benzie IF. Vitamin C: prospective functional markers for defining 2 Lene Frost Anderson, unpublished data. optimal nutritional status. Proc Nutr Soc 1999;58:469-76.

Cancer Epidemiol Biomarkers Prev 2004;13(5). May 2004

18. Drewnowski A, Rock CL, Henderson SA, et al. Serum h-carotene 31. Naska A, Vasdekis VG, Trichopoulou A, et al. Fruit and vegetable and vitamin C as biomarkers of vegetable and fruit intakes in a availability among ten European countries: how does it compare community-based sample of French adults. Am J Clin Nutr 1997;65: with the ‘five-a-day’ recommendation? DAFNE I and II projects of 1796-802. the European Commission. Br J Nutr 2000;84:549-56. 19. Borel P, Grolier P, Mekki N, et al. Low and high responders to 32. Johansson L, Andersen LF. Who eats 5 a day?: intake of fruits and pharmacological doses of h-carotene: proportion in the population, vegetables among Norwegians in relation to gender and lifestyle. mechanisms involved and consequences on h-carotene metabolism. J Am Diet Assoc 1998;98:689-91. J Lipid Res 1998;39:2250-60. 33. Almendingen K, Trygg K, Pedersen JI. An assessment of the use of 20. Parker RS, Swanson JE, You CS, Edwards AJ, Huang T. Bioavailabil- simple methods to predict individual energy intakes for intervention ity of carotenoids in human subjects. Proc Nutr Soc 1999;58:155-62. studies. Eur J Clin Nutr 1998;52:54-9. 21. Roodenburg AJ, Leenen R, het Hof KH, Weststrate JA, Tijburg LB. 34. Energy and protein requirements. Geneva: WHO; 1985. Amount of fat in the diet affects bioavailability of lutein esters but 35. Altman DG. Practical statistics for medical research. London: not of a-carotene, h-carotene, and vitamin E in humans. Am J Clin Chapman and Hall/CRC; 1991. Nutr 2000;71:1187-93. 36. Pocock SJ. Clinical trials; a practical approach. London: John Wiley 22. Brady WE, Mares-Perlman JA, Bowen P, Stacewicz-Sapuntzakis M. and Sons; 1983. Human serum carotenoid concentrations are related to physiologic 37. Redmond CK, Colton T. Biostatistics in clinical trials. Chichester: and lifestyle factors. J Nutr 1996;126:129-37. Wiley; 2001. 23. Kitamura Y, Tanaka K, Kiyohara C, et al. Relationship of alcohol use, 38. Kawaii S, Tomono Y, Katase E, Ogawa K, Yano M. Quantitation physical activity and dietary habits with serum carotenoids, retinol of flavonoid constituents in citrus fruits. J Agric Food Chem 1999;47: and a-tocopherol among male Japanese smokers. Int J Epidemiol 3565-71. 1997;26:307-14. 39. USDA Database for the Flavonoid Content of Selected Foods. 24. Olmedilla B, Granado F, Blanco I, Rojas-Hidalgo E. Seasonal and sex- U.S.D.A. Agricultural Research Service; 2003. related variations in six serum carotenoids, retinol, and a-tocopherol. 40. Hollman PC, van Trijp JM, Buysman MN, et al. Relative bioavail- Am J Clin Nutr 1994;60:106-10. ability of the antioxidant flavonoid quercetin from various foods in 25. Rock CL, Flatt SW, Wright FA, et al. Responsiveness of carotenoids man. FEBS Lett 1997;418:152-6. to a high vegetable diet intervention designed to prevent breast can- 41. Hollman PC, Katan MB. Dietary flavonoids: intake, health effects and cer recurrence. Cancer Epidemiol Biomark Prev 1997;6:617-23. bioavailability. Food Chem Toxicol 1999;37:937-42. 26. Alberg A. The influence of cigarette smoking on circulating 42. Perez-Vizcaino F, Ibarra M, Cogolludo AL, et al. Endothelium- concentrations of antioxidant micronutrients. Toxicology 2002;180: independent vasodilator effects of the flavonoid quercetin and its 121-37. methylated metabolites in rat conductance and resistance arteries. 27. Nielsen SE, Freese R, Kleemola P, Mutanen M. Flavonoids in human J Pharmacol Exp Ther 2002;302:66-72. urine as biomarkers for intake of fruits and vegetables. Cancer Epi- 43. Aziz AA, Edwards CA, Lean ME, Crozier A. Absorption and demiol Biomark Prev 2002;11:459-66. excretion of conjugated flavonols, including quercetin-4V-O-h-gluco- 28. Nielsen SE, Freese R, Cornett C, Dragsted LO. Identification and side and isorhamnetin-4V-O-h-glucoside by human volunteers after quantification of flavonoids in human urine samples by column- the consumption of onions. Free Radic Res 1998;29:257-69. switching liquid chromatography coupled to atmospheric pressure 44. Young JF, Nielsen SE, Haraldsdottir J, et al. Effect of fruit juice chemical ionization mass spectrometry. Anal Chem 2000;72:1503-9. intake on urinary quercetin excretion and biomarkers of antioxida- 29. Erlund I, Meririnne E, Alfthan G, Aro A. Plasma kinetics and urinary tive status. Am J Clin Nutr 1999;69:87-94. excretion of the flavanones naringenin and hesperetin in humans after 45. Agudo A, Slimani N, Ocke MC, Naska A. Vegetable and fruit ingestion of orange juice and grapefruit juice. J Nutr 2001;131:235-41. consumption in the EPIC cohorts from 10 European countries. IARC 30. Nielsen SE, Young JF, Daneshvar B, et al. Effect of parsley (Petro- Sci Publ 2002;156:99-103. selinum crispum) intake on urinary apigenin excretion, blood anti- 46. Fuhr U, Kummert AL. The fate of naringin in humans: a key to oxidant enzymes and biomarkers for oxidative stress in human grapefruit juice-drug interactions? Clin Pharmacol Ther 1995;58: subjects. Br J Nutr 1999;81:447-55. 365-73.

Cancer Epidemiol Biomarkers Prev 2004;13(5). May 2004

Downloaded from cebp.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. Urinary Excretion of Flavonoids Reflects Even Small Changes in the Dietary Intake of Fruits and Vegetables

Asgeir Brevik, Salka Elbøl Rasmussen, Christian A. Drevon, et al.

Cancer Epidemiol Biomarkers Prev 2004;13:843-849.

Updated version Access the most recent version of this article at: http://cebp.aacrjournals.org/content/13/5/843

Cited articles This article cites 36 articles, 14 of which you can access for free at: http://cebp.aacrjournals.org/content/13/5/843.full#ref-list-1

Citing articles This article has been cited by 1 HighWire-hosted articles. Access the articles at: http://cebp.aacrjournals.org/content/13/5/843.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cebp.aacrjournals.org/content/13/5/843. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.