Cancer Epidemiology, Biomarkers & Prevention 843 Urinary Excretion of Flavonoids Reflects Even Small Changes in the Dietary Intake of Fruits and Vegetables 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 Downloaded from cebp.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. 844 Biomarkers for the Intake of Fruits and Vegetables 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.