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* Institute of Food Safety and Nutrition, Danish Veterinary and Food Administration, DK-2860 Søborg, Denmark and
Department of Human Nutrition, Royal Veterinary and Agricultural University, DK-1958 Frederiksberg, Denmark
2To whom correspondence should be addressed. E-mail: salka{at}fdir.dk.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • biomarkers • flavonoids • fruits and vegetables • diet-controlled • human intervention
A high intake of fruits and vegetables has been associated with a reduced risk of coronary heart disease (1–4) and certain types of cancer (5–7). In epidemiologic studies on the association between diet and disease, dietary intake data are often based on self-reported data, which carries a risk for subjective bias (8–11). Furthermore, consumption of healthy foods such as fruits and vegetables may be overreported (12). Dietary biomarkers represent an objective alternative to the traditional food registration methods. Flavonoids are widely distributed in fruits and vegetables regularly consumed by humans and are thus a potential biomarker for the dietary intake of fruits and vegetables. We recently reported that 7 dietary flavonoids could be determined simultaneously in urine by a sensitive and analytically validated method (13), and that the 24-h urinary excretion of these flavonoids may have potential as a new biomarker for fruit and vegetable intakes (14,15). These studies demonstrated that high intakes of fruits and vegetables are easily detected by the flavonoid biomarker, both in subjects consuming controlled diets and in those consuming their habitual diet (14,15). An essential property of a biomarker in studies of diet-disease associations is that the biomarker changes in a linear manner with varying exposures. It is therefore important to further validate the flavonoid biomarker and to evaluate its responsiveness in intervention studies with more moderate changes in fruit and vegetable intake. In the present study, we therefore investigated the 24-h urinary excretion of 7 dietary flavonoids, including the flavonols quercetin, kaempferol, isorhamnetin, tamarixetin, the flavanones hesperetin and naringenin, and the dihydrochalcone, phloretin (Fig. 1), after 0, 300, and 600 g/d of fruit and vegetable intake. Because collection of complete 24-h urine samples is both difficult and time-consuming, we investigated further whether a morning urine sample could substitute for the collection of 24-h urine samples.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Study design and diet. Each volunteer participated in two 5-d periods; d 1–3 of each period were "run-in-days" in which subjects followed a self-restricted diet without any fruits, vegetables, potatoes, red wine, tea, or coffee. On d 4 of each period, the men were provided a strictly controlled diet containing no fruits or vegetables (basic diet). On d 5 of each period, they consumed the basic diet supplemented with either 300 g of fruit and vegetable mixture (low FVDiet) or 600 g fruit and vegetable mixture (High FVDiet) in random order. On d 4 and 5, breakfast and lunch were provided at the Department of Human Nutrition between 0715 and 0735 h and 1215 and 1245 h, respectively. The dinner meal provided was consumed at home between 1800 and 2000 h. The basic diet contained no fruits and vegetables and was based on bread, meat, pasta, rapeseed oil, butter, and low-fat milk. The fruit and vegetable diet included items commonly eaten in Denmark (17) (Table 1). The menus were prepared in advance and frozen at -18°C until use. With a few exceptions (white bread and low-fat milk), all of the ingredients were from the same batch. All of the men received the same energy level. The differences in energy intake among the 3 types of diets were equalized with a drink rich in carbohydrates. The energy intake and the concentration of macronutrients in the diet were calculated with Dankost2000 software based on the Danish Veterinary and Food administration food composition database; the concentrations of flavonoids, potassium, and vitamin B-6 in the diet were based on chemical analysis (see Table 2). The study was approved by the Ethics Committee of Copenhagen and Frederiksberg municipality (J. No. KF01–161/01). Informed written consent was obtained from all participants before the study.
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Determination of urinary flavonoids.
The 7 dietary flavonoids (Fig. 1) were determined in urine by liquid chromatography-mass spectrometry (LC-MS) using the method of Nielsen et al. (13) with small alterations with respect to the internal standards used. In short, to 2-mL aliquots of each urine sample were added 500 ng of 3X 13C isotopic labeled genistein and 500 ng of genistein as an internal standard [20 ng/µL dimethyl sulfoxide (DMSO)] and hydrolyzed enzymatically as previously described (13). The hydrolyzed samples were evaporated completely under vacuum and redissolved in 10% aqueous methanol and 1% formic acid. Then a DMSO stock solution containing 500 ng of 3X 13C-daidzein and 500 ng of daidzein was added as an additional internal standard (20 ng/µL DMSO). The samples were then centrifuged at 11000 x g for 10 min at 20°C and the entire amount of the supernatant (
300 µL) was injected into the LC-MS system. The flavonoid concentrations in the urine samples were determined as single determinations based on calibration curves generated by spiked blank urine samples as described previously (13). In addition, supplementary calibration samples were included at the beginning, during, and at the end of each series of samples, and a final adjustment was performed based on the level of the internal standards in each sample. Before and after each series of samples, the performance of the entire LC-MS assay was controlled by injections of aliquots containing all of the employed flavonoid standards, including the internal standards. The enzymes used for the enzymatic hydrolysis of the flavonoid glycosides in the urine samples were arylsulphatase (Aerobacter aerogenes, 16.8 standard kU/L) from Sigma Chemical and ß-glucoronidase (Escherichia coli, >200 standard kU/L) obtained from Boehringer Mannheim. Methanol and acetonitrile were of HPLC grade and obtained from Rathburne. The flavonoid standards, quercetin and naringenin, were obtained from Aldrich. Kaempferol, isorhamnetin, tamarixetin, genistein, and daidzein were obtained from Apin Chemicals. Phloretin and hesperetin were purchased from Sigma Chemical. All standards were of HPLC grade. A stock solution of 100 ng/µL of a mixture of all of the flavonoid aglycone standards was prepared in DMSO. Stock solutions of 20 ng/µL of the internal standards 3X 13C genistein, genistein, 3X 13C-daidzein and daidzein were prepared in DMSO. The isotopically labeled internal standards, 3X 13C genistein, and 3X 13C-daidzein were obtained from the School of Chemistry, University of St. Andrews, United Kingdom. All stock solutions were stored at -20°C and were stable for at least 3 mo.
Determination of urinary 4-PA. The urinary concentration of 4-PA was determined by HPLC (26). The interassay CV for 4-PA was 2.2% (n = 16).
Determination of urinary potassium. Potassium was determined by flame photometry as described by Boling (25). Analysis was done with the Ciba-corning flame photometer 410. Calibration of the instrument was performed before analysis with 1 mL diluent concentrate (Sherwood, Cat/Best. No. 011 56 681) in 1 L deionized water. For every 20 samples, an internal standard [Multical TM 200Na/100K/45CO2 (Bayer, commissions no. 478524), Bio-Rad] and an external standard [Lyphocheck 174Na/110K, Quantitative Urine Control (abnormal), Bio-Rad 377, 2 (lot. no. 60152)] were analyzed. The interassay CV for potassium was 1.7% (n = 22).
Chemical analysis of the diet. The concentration of flavonoids in the fruit and vegetable supplements was determined after acid hydrolysis by HPLC and MS as described by Justesen et al. (27), with the exception of the compound phloridzin. Phloridzin was determined by HPLC and MS without prior hydrolysis, after extraction and solid phase extraction (SPE) cleaning by the method described by Grinder-Pedersen et al. (28), which filters the extract through a 5-µm filter before SPE. The SPE eluate was filtered through a 0.45-µm filter before HPLC, which was performed on a Jupiter Phenomenex RP C18 column (250 x 4.6 mm, 5 µm). The mobile phase consisted of 1% formic acid:99% water (A), 23% acetonitrile:23% methanol:1% formic acid:47% water (B) and 100% methanol (C). The gradient was 100% A to 100% B in 21 min,100% B to 100% C from 21 to 26 min, and 100% C from 26 to 38 min. Phloridzin was quantified at 289 nm. The concentration of flavonoids in the basic diet was determined by the method of Grinder-Petersen et al. (28). The concentration of potassium was determined by inductively coupled plasma-atomic emission spectroscopy after high-pressure wet-ashing of the samples with nitric acid in polytetrafluoroethylene-lined steel bombs (internal method FM.061.1). Potassium quantification was done by external calibration and in all analytical series, a sample of certified reference material and a blank were included to ensure analytical quality. The potassium concentration was determined as duplicates. The concentration of vitamin B-6 in the diet was determined by HPLC using the method of Kall et al. (29).
Statistical analysis. The statistical analyses were performed using the SPSS package program version 10.0. P-values < 0.05 (two-tailed) were considered to be significant. Variables were examined for normality and skewness and were tested for carry-over and treatment-period interactions. The concentrations of flavonoids, 4-PA, and potassium in 24-h and morning urine samples were not normally distributed, and because logarithmic transformation did not normalize them, nonparametric tests were used in the statistical analysis of the data (30). Wilcoxon matched pairs tests were performed to compare the urinary excretion of the selected biomarkers at the different intake levels of fruits and vegetables. Spearman’s correlation (rs) was used to examine associations between the intake level of fruits and vegetables and the excretion of biomarkers in urine.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Flavonoids. The mean 24-h urinary excretion of all the individual flavonoids and the sum of all 7 flavonoids determined in urine (total flavonoids) increased significantly with increased fruit and vegetable intake (see Table 3). Furthermore, there was a strong linear correlation between the mean excretion of total flavonoids and the fruit and vegetable dose in 24-h urine (rs = 0.86, P < 1 x 10-6 (Fig. 2A). Most of the individual flavonoids tested had a similar positive and significant correlation with the intake of fruits and vegetables in 24-h urine (rs between 0.73 and 0.83, P < 1 x 10-5) except for phloretin (rs = 0.28, P = 0.1). Among the 12 men, two were high-excreters with a flavonoid excretions > 2 times the mean excretion of the other 10 men (Fig. 2A).
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To investigate the linear responsiveness of the individual flavonoids to the dietary treatments, the excreted fraction of the two different flavonoid doses was calculated (see Table 3). Ideally, the fraction excreted in urine should be independent of the given flavonoid dose, and this was in general the case in the 24-h urine sample. The citrus flavonoids, hesperetin and naringenin, however, had a different excretion profile, and the fraction of hesperetin excreted was 10 times higher than the investigated flavonols (Table 3); in addition, the excretion of naringenin and phloretin exceeded the excretion of the flavonols. In general, a much lower fraction of the flavonoid dose was found in the morning urine.
4-PA and potassium. The urinary excretion of the vitamin B-6 metabolite 4-PA was used as a measure of the intake of vitamin B-6. We found that the excretion of 4-PA in 24-h and morning urine samples increased significantly only with the 600 g increase in the fruit and vegetable intake, whereas an increase of 300 g did not affect 4-PA excretion (see Table 3).
Because the excretion of potassium in morning urine on d 4 of the first crossover period of the study was lower than that on d 4 of the second crossover period (P = 0.003, see Table 3), the potassium data could not be combined. Instead, Wilcoxon matched-pairs tests were performed to investigate the response to the fruit and vegetable supplementation in relation to the treatment order. However, the changes in the fruit and vegetable intake employed in the present study did not affect the excretion of potassium in urine (Table 3).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In the present study, the 24-h urinary excretion as a percentage of intake of total flavonoids and the majority of the individual flavonoids was independent of the flavonoid dose, demonstrating that the urinary excretion of these flavonoids reflects the dietary intake in a dose-dependent manner, within the dosage range tested. It was shown previously that the flavonoids excreted in 24 h reflect the preceding flavonoid dosages (14); however, more information on the dose dependency is required. The excretion levels observed in the present study are in the range of previous observations, although there are great variations reported in the literature. Earlier studies reported excretion levels in humans between 4.1 and 24.4% for hesperetin, 1.1–30.2% for naringenin 0.2–1.4% for quercetin, and 0.9–2.5% for kaempferol (31–38). More studies are thus warranted to explain these large variations in the fractions excreted in urine.
Because the flavanones and dihydrochalcones are excreted to a greater extent than the flavonols, they may also be absorbed to a greater extent than the flavonols and thus have an increased potential of exerting biological activity, thereby preventing diseases. On the other hand, the relatively longer plasma half-life of the flavonol quercetin (15.1–28.0 h) (32,39) in relation to the flavanones (1.3–2.9 h) (31,40) may diminish the biological importance of these flavonoids. Further studies are warranted, however, before final conclusions on the biological importance of these observations can be made.
All 7 flavonoids employed in the analytical set-up were measurable in the urine. A recent study suggests, however, that some of the phloretin present in urine could be a result of endogenous metabolism of naringenin to phloretin (41). To our knowledge, few reports exist on the excretion of phloretin in human urine (14,15,42). In the present study, 5.5% of the phloretin dose was excreted in the 24-h urine. In a study performed by DuPont and co-workers (42) with alcoholic apple cider, as much as 21% of the phloretin dose was excreted in 24-h urine. In the present study, the correlation between the urinary excretion of phloretin and the fruit and vegetable intake was not significant. The lack of a correlation may be due to the rather low amount of phloretin consumed in the present study (0.4 or 0.8 mg/d) and to the high variation in the urinary phloretin data. The amount of apple consumed in the present study (40 or 80 g/d) was low compared with the average weight of an apple (150 g); thus, urinary phloretin may be sensitive enough to reflect higher and more realistic apple intakes.
The presence of tamarixetin in urine is probably a result of endogenous metabolism of quercetin catalyzed by catechol-O-methyltransferase, which results in the formation of isorhamnetin and tamarixetin by monomethylation of the B-ring (43,44). Tamarixetin was positively affected by the fruit and vegetable intervention; thus, the effect of this endogenous metabolism apparently increases with increasing quercetin intake.
Even though the study group in the present study was relatively homogenous and consumed a standardized diet, there was a high interindividual variation in the amount of flavonoids excreted in urine (Table 3). This was caused mainly by 2 high-excreters, with flavonoid excretion 2–3 times the mean of the other 10 men (see Fig. 2A). Exclusion of the 2 high-excreters from the data analysis did not considerably affect the correlation coefficients between the fruit and vegetable intake and the excretion of total flavonoid in 24-h or morning urine (rs = 0.89, P < 1 x 10-6 and rs = 0.56, P < 0.001, respectively). Because of the high interindividual variation in the excretion of the flavonoids, the flavonoid biomarker is not a particularly precise biomarker for intake levels of fruits and vegetables at the individual level. The high interindividual variation in the flavonoid excretion and the presence of a few "high-excreters" agrees with observations in previous studies (14,31,40,45). In contrast, the intraindividual variation was low, which also agrees with previous studies (37,38). Because of the low intraindividual variation, the excretion of flavonoids in 24-h urine has great potential as a compliance biomarker and as a biomarker for both controlled and uncontrolled changes in the intake level of fruits and vegetables achieved by an intervention.
In the present study, we also determined the concentration of 4-PA and potassium in the urine samples collected to investigate whether these compounds reflected changes in the fruit and vegetable intake. The present study showed, however, that the responsiveness of 4-PA was insufficient to serve as a biomarker of fruit and vegetable consumption levels because the increase in the fruit and vegetable intake did not result in a linear response in the excretion of 4-PA in 24-h or morning urine. Potassium also did not reflect the changes in the intake level of fruits and vegetables. According to Jacobsen et al. (46) and Bingham et al. (47), the amount of potassium excreted in the urine is dependent on the amount of fiber consumed. Thus, increased fiber consumption results in increased excretion of vitamin B-6 in the feces. Because fruits and vegetables are a rich source of fiber, it is possible that the lack of an association between fruit and vegetable intake and potassium excretion in the present study could be explained in part by the higher fiber intake. Further studies with feces collection or fiber-adjusted diets are required before such a conclusion can be drawn.
In conclusion, the present study demonstrated that the excretion of flavonoids in 24-h urine could be used as a new biomarker for fruit and vegetable intake, and that it is useful also for lower intake levels. Flavonoids in morning urine samples responded to the different fruit and vegetable doses in a nonlinear manner and are therefore not useful for detecting modest changes in dietary intakes of fruits and vegetables. The other urinary markers investigated, 4-PA and potassium, are not applicable as quantitative biomarkers of fruit and vegetable intake.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: DMSO, dimethyl sulfoxide; FVDiet, fruit and vegetable diet; LC-MS, liquid chromatography-mass spectrometry; 4-PA, 4-pyridoxic acid; rs, Spearman’s correlation coefficient; SPE, solid phase extraction.
Manuscript received 3 September 2003. Initial review completed 16 October 2003. Revision accepted 18 November 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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