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Human Nutrition and Metabolism

The Relative Bioavailability of Enterolignans in Humans Is Enhanced by Milling and Crushing of Flaxseed¹

Anneleen Kuijsten^*,, Ilja C. W. Arts^*, Pieter van’t Veer and Peter C. H. Hollman^*,2

^* RIKILT-Institute of Food Safety, Wageningen UR, 6700 AE, Wageningen, The Netherlands and Wageningen University, Food Technology and Nutritional Sciences, Division of Human Nutrition, 6700 EV, Wageningen, The Netherlands

²To whom correspondence should be addressed. E-mail: peter.hollman{at}wur.nl.

ABSTRACT

Flaxseed is one of the richest sources of lignans and is increasingly used in food products or as a supplement. Plant lignans can be converted by intestinal bacteria into the so-called enterolignans, enterodiol and enterolactone. For a proper evaluation of potential health effects of enterolignans, information on their bioavailability is essential. The aim of this study was to investigate whether crushing and milling of flaxseed enhances the bioavailability of enterolignans in plasma. In a randomized, crossover study, 12 healthy subjects supplemented their diet with 0.3 g whole, crushed, or ground flaxseed/(kg body weight · d). Each subject consumed flaxseed for 10 successive days separated by 11-d run-in/wash-out periods, in which the subjects consumed a diet poor in lignans. Blood samples were collected at the end of each run-in/wash-out period, and at the end of each supplement period. Plasma enterodiol and enterolactone were measured using LC-MS-MS. The mean relative bioavailability of enterolignans from whole compared with ground flaxseed was 28% (P 0.01), whereas that of crushed compared with ground flaxseed was 43% (P 0.01). Crushing and milling of flaxseed substantially improve the bioavailability of the enterolignans.

KEY WORDS: • bioavailability • enterodiol • enterolactone • lignans • flaxseed

Lignans are biphenolic compounds that occur in foods of plant origin. Some plant lignans can be converted into the enterolignans, enterodiol and enterolactone, through a series of reactions mediated by the bacterial flora in the colon (1,2). Enterolignans become available in the blood circulation 8–10 h after ingestion of plant lignans (3–5). They are eliminated slowly (5) and excreted via urine and feces (3–12).

Two epidemiologic studies suggested that high serum concentrations of enterolactone are associated with a lower risk of acute coronary events (13,14). Associations between enterolignans and cancer are less clear. Inverse associations for breast cancer were reported in case-control studies, and one prospective study (15–18), whereas no associations were found in most prospective studies (19–23).

Among foods consumed by humans, flaxseed contains the highest concentration of enterolignan precursors, mainly secoisolariciresinol diglucoside. Other seeds, nuts, whole grains, fruits and vegetables, and beverages such as coffee and tea contain smaller amounts (24). The most important sources of lignan precursors in Western diets are beverages such as tea and coffee, seeds, cereals, berries, fruits and vegetables (25,26). Flaxseed is a relatively minor dietary component in most countries, but because of its potential health benefits [flaxseed also contains a high quantity of (n-3) fatty acids as well as dietary fiber], it is increasingly being incorporated into a variety of food products, such as bread, muesli bars, and breakfast cereals, or used as a supplement. For example, in the Netherlands, whole flaxseed is used in commercial breads (up to 3.5 g flaxseed/100 g bread); therefore is an important potential source of dietary lignans. In a case-control study carried out in Texas, Strom et al. (27) found that flaxseed bread was one of the main food sources of lignans.

Flaxseed is a small hard-coated seed. Whole seeds are used in breads, whereas most supplements consist of crushed seeds. We questioned whether lignans in whole flaxseeds are accessible to bacteria in the colon. We expected that milling or crushing would substantially enhance the accessibility of the bacteria to the plant lignans, and as a result, would improve their conversion into enterolignans. In the present study, we investigated whether milling and crushing enhanced the bioavailability of enterolignans from flaxseed.

SUBJECTS AND METHODS

    Subjects. Six men and 6 women, with a mean age of 25 (range 18–64) y, participated in this study. Subjects were recruited in Wageningen by advertisement in the university newspaper, by flyers, and by posters. All subjects were generally healthy (self-reported). None of the subjects had diarrhea, or had used antibiotics or other medication in the past 3 mo, except for oral contraceptives or analgesics. Height and weight were measured with the participants wearing indoor clothing and no shoes. The men weighed (mean ± SD) 72.3 ± 5.2 kg, and the women weighed 59.2 ± 6.7 kg. The BMI in men was 22.0 ± 1.7 kg/m², and 19.9 ± 1.2 kg/m² in women. During the study, subjects did not lose or gain weight. Subjects were excluded if their hemoglobin concentration was low (<120 g/L for women and <140 g/L for men) or if their urine contained traces of glucose or protein (test strip for rapid determination of glucose and protein in urine, Macherey-Nagel). Vegans, vegetarians (defined as persons who consume fish or meat <1 time/wk), and people consuming flax-containing supplements were excluded, as were pregnant or lactating women. The Medical Ethical Committee of the Department of Human Nutrition at Wageningen University (The Netherlands) approved the study, and all subjects gave their informed consent. All subjects completed the study.

    Experimental design. This study was designed as a randomized, crossover trial consisting of three 10-d supplement periods preceded by three 11-d run-in/wash-out periods. During the supplement periods, subjects consumed whole, crushed, or ground flaxseed provided by us. To avoid interference from other dietary sources of lignans, volunteers consumed a diet poor in lignans (see below) throughout the entire study. On d 10 of the supplement periods and d 11 of the run-in/wash-out periods, 3 venous blood samples were drawn from each subject. Men and women were randomly allocated to 1 of 6 treatment order groups so that each group consisted of 1 man and 1 woman.

    Diet. To avoid interference from other dietary sources of lignans, the participants consumed a diet poor in lignans (contributing <5% of the intake from the flaxseed supplements) throughout the entire study. Food products were classified (allowed, limited, or to be avoided) according to the amount consumed, and to the lignan content of the foods (24). The participants were given a list with the classified food products. They avoided several fruits (e.g., dried fruits, berries, peach, pear, nectarine, and apricot), a few vegetables (e.g., cabbage, broccoli, and haricot beans), seeds and nuts (e.g., flaxseed, sesame seed, and peanut), breakfast cereals, cereal and muesli bars, whole-grain products (e.g., rye bread and whole grain bread), and herbal tea. Furthermore, they limited their daily consumption of several fruits (e.g., kiwi, orange, and cherries) to 100–150 g, tea to 300 mL or coffee to 800 mL, fruit juices to 660 mL, and beer to 800 mL or wine to a maximum of 300 mL. Consumption of selected wheat products (white bread, pasta), rice, milk products (milk, yogurt, and cheese), meat and fish, several fruits (e.g., apple, grapes, and banana) and vegetables (e.g., cucumber, tomatoes, leek, and spinach) was allowed so that, in principle, the intake of micro- and macronutrients was adequate. To ensure an adequate fiber intake, wheat bread with a low lignan content (370 nmol lignans/100 g bread) was supplied daily. Bread is an important source of fiber in The Netherlands. When subjects accidentally consumed too much of a specific food product they were instructed to report the time of eating and the amount eaten of this specific product in a diary.

    Supplements. Flaxseed was industrially pretreated with steam to reduce microbiological contamination. Whole and crushed flaxseeds were prepared from the same batch and were provided by a commercial retailer (Weller-Verhoef). To obtain ground flaxseed, we milled part of the whole flaxseed to a coarse texture using a household blender (Waring) for 10 s 4 times and homogenized the particles. To determine the lignan contents of the flaxseed supplements, secoisolariciresinol, matairesinol, pinoresinol, and lariciresinol were measured by LC-MS-MS (28). The flaxseed contained 3.6 mg lignans/g (>98% consisted of secoisolariciresinol); the whole, crushed, and ground flaxseeds did not differ.

During the supplement periods, subjects consumed 0.3 g flaxseed/(kg body weight · d) (3.0 µmol lignans/kg body weight). They consumed the flaxseed supplements in 2 servings, at breakfast and dinner, mixed with applesauce or custard sauce. On d 10 of the supplement periods, when blood samples were taken, the subjects consumed only 1 serving of flaxseed at breakfast. Before the study, each flaxseed serving was weighed, packed individually, and stored at –20°C. Subjects received the packages twice a week, with instructions to keep the flaxseed frozen until consumption. Subjects were instructed to consume the entire contents of the individual packages. Furthermore, subjects were not allowed to cook or microwave the flaxseed. Used packages were collected to monitor compliance. In addition, subjects were asked to record the time at which they consumed each flaxseed supplement.

    Collection of samples. On d 10 of each supplement period and on d 11 of each run-in/wash-out period, 3 venous blood samples were taken into vacuum tubes containing EDTA. The first sample was taken after a 12-h fast between 0700 and 0800 h. The second sample was taken immediately before lunch between 1200 and 1300 h, and the third sample was taken immediately before dinner between 1700 and 1800 h. Samples were centrifuged within 30 min at 1187 x g for 10 min at 4°C and stored at –80°C until analysis.

    Analytical methods. The concentrations of enterodiol and enterolactone in plasma were measured by LC-MS-MS using triply ¹³C-labeled isotopes (29). Briefly, enterolignans were measured in 300 µL plasma after hydrolysis of conjugates using a freshly prepared enzyme mixture of ß-glucuronidase-sulfatase from Helix pomatia at 37°C for 4 h. Subsequently, samples were extracted twice with diethyl ether. The ether fractions were combined and transferred into new tubes containing 40% methanol:water (v:v). The ether fraction was evaporated under a gentle stream of nitrogen at 30°C. Before the analysis, extracts were filtered, transferred into vials, and injected into the LC-MS system. The limit of detection was 0.15 nmol/L for enterodiol and 0.55 nmol/L for enterolactone. All plasma samples from 1 subject were analyzed in 1 assay. Laboratory technicians were unaware of a subject’s treatment group.

    Statistical analysis. The individual mean enterolignan plasma concentrations on each day were based on the 3 blood samples taken. The relative bioavailability for each individual was defined as the ratio of the mean plasma enterolignan concentration after the supplement periods and the mean plasma enterolignan concentration after supplementation with ground flaxseed (reference) multiplied by 100. The plasma enterolignan concentrations at the end of each supplement period were corrected for the baseline value, which was based on the mean concentration of all run-in/wash-out periods. The differences between the supplement periods were tested by paired t tests. The effects of treatment order per supplementation group (whole, crushed, or ground flaxseed) were tested with one-way ANOVA. Differences were considered significant at P 0.05, unless stated differently. Estimates of within-subject and between-subject variances for each supplement period were obtained by the SPSS reliability command using 1-way ANOVA. The within-subject and between-subject CV were calculated as the square root of the variance component estimates (²_b and ²_w) divided by the overall mean multiplied by 100, where ²_b is the between-subject variance component and ²_w is the within-subject variance component. The reproducibility of measurements in plasma, an important characteristic for a biomarker, was evaluated using the intraclass correlation coefficient (ICC). The ICC represents the proportion of variance in the measure explained by the between-subject variation, i.e., ICC = ²_b/(²_b + ²_w). High ICC values (close to 1) represent excellent reproducibility. The ICC of a single measurement was estimated assuming a 1-way random effects model. All statistical analyses were performed with SPSS statistical software package (version 10.0). Data are presented as means ± SEM.

RESULTS

The highest concentrations of enterolignans were reached after supplementation with ground flaxseed (range 122–539 nmol/L) (Table 1). Plasma enterolignan concentrations after supplementation with crushed (22–277 nmol/L), and whole flaxseed (29–262 nmol/L) were lower. We found no significant effect of treatment order. The relative bioavailability of enterolignans from whole flaxseed was 28% (range 14–78%), whereas the relative bioavailability of enterolignans from crushed flaxseed was 43% (14–83%; Fig. 1); both differed significantly from ground flaxseed and from one another. Men and women did not differ in relative bioavailability. Because the plasma concentrations of enterolignans within subjects did not differ among the 3 run-in/wash-out periods, we used the mean concentration of all 3 run-in/wash-out periods per subject to calculate their mean baseline concentration. One of the subjects had an unexplained high concentration of enterodiol (61 nmol/L) and of enterolactone (74 nmol/L) at the end of the first run-in/wash-out period. We excluded this value because at the end of the other run-in/wash-out periods, the concentrations of both enterolignans in this subject were much lower (enterodiol: 0.0 and 0.4 nmol/L; enterolactone 3.5 and 13.8 nmol/L). The overall mean baseline concentration of enterodiol in plasma was 1.9 ± 0.5 nmol/L and of enterolactone 9.5 ± 1.1 nmol/L (Table 1).

View larger version (17K): [in this window] [in a new window]   FIGURE 1 Relative bioavailability in human subjects of lignans from whole and crushed flaxseed compared to ground flaxseed (set at 100%). Values are means ± SEM, n = 12. aDifferent from crushed flaxseed (P 0.05); bdifferent from ground flaxseed, P 0.01.

View larger version (13K): [in this window] [in a new window]   FIGURE 2 Contribution of enterolactone and enterodiol (%) to the total concentration of enterolignans in plasma of human subjects per supplement period. Values are means ± SEM, n = 12. aDifferent from ground flaxseed, P 0.05.

View larger version (18K): [in this window] [in a new window]   FIGURE 3 Plasma concentrations of enterodiol (A) and enterolactone (B) in human subjects at various times during the day after the supplement period. Values are means ± SEM, n = 12. aDifferent from the first sample (0700–0800 h); bdifferent from the second sample (1200–1300 h).

DISCUSSION

In a randomized crossover study, we showed for the first time that crushing and milling of flaxseed improved the bioavailability of lignans 2- to 4-fold compared with whole flaxseed. This increased bioavailability is likely due to the improved accessibility of the colon bacteria to crushed and ground flaxseed.

In our study, the total plasma enterolignan concentration in subjects consuming 15–25 g ground flaxseed for 10 d (270 nmol/L) was higher than in Finnish subjects consuming 25 g ground flaxseed for 8 successive d (85 nmol/L) (3). A simple explanation for this difference is the lignan content of the flaxseed supplements, which was >2 times higher in our study (9.91 µmol/g) than in the Finnish study (2.97 µmol/g).

Steady-state plateau concentrations will be reached when the supplemental interval (10–14 h in this study) is smaller than 4 times the elimination half-life (4.4 h for enterodiol, 12.6 h for enterolactone) (30), which was true in our study. The elimination half-life of 12.6 h predicts that steady state will be reached after 2 d (4 x 12.6 h); thus the 10 d of supplementation were sufficient. Nevertheless, fluctuations in enterolignan plasma concentrations are to be expected because of the noncontinuous dosing. To improve accuracy, 3 blood samples were taken on 1 d to measure the enterolignan concentrations. In spite of the diet restrictions and the standardized intake of flax supplements, there were large variations within (24–38%) and between subjects (46–98%). The within-subject variation was similar to that (31%) in 6 postmenopausal women consuming an experimental diet with 142 µg lignans/d (31). Higher within-subject variations were observed over periods of weeks (67%) and months (68%) among 20 Finnish men and women consuming their normal diet (median concentration of enterolactone was 9.1 nmol/L) (32). The ratio of within-subject and between-subject variation in our study was quite low, resulting in high ICCs for enterodiol (0.86–0.95) and enterolactone (0.57–0.84). Even at low concentrations, the ICC was relatively high (0.56–0.72; Table 2). Our results indicate that a single measurement will accurately reflect an individual’s mean short-term concentration. Note, however, that this result was obtained in a well-controlled trial with standardized consumption of flaxseed. Moderately high ICCs of plasma enterolactone measurements were observed over a period of weeks (0.79) and months (0.77) in men and women consuming their normal diet (32). As expected, lower ICCs of serum measurements (enterolactone: 0.55; enterodiol 0.37) were observed over a 2-y period (33). Our results and those of others indicate that plasma enterolactone and enterodiol are relatively good biomarkers. Although there was a relatively high within-subject variation, distinction between individuals is feasible as indicated by the relatively high ICC.

When we studied the within-subject variation in more detail, we observed that the daily variation of enterolactone was higher than that of enterodiol (Fig. 3). This was surprising because the elimination half-life of enterolactone, obtained in a single-dose experiment, was longer than that of enterodiol (5), which would predict less variation (30). It is possible that absorption and elimination are affected differently by enterodiol and enterolactone during chronic supplementation, leading to changes in elimination. Alternatively, the daily meal pattern may have influenced the availability of enterolignans. We observed that enterolactone concentrations were higher in the morning than late in the afternoon (Fig. 3). In the morning, subjects consumed flaxseed supplements with breakfast (light meal), and in the evening with dinner (heavy meal). When flaxseed supplements are consumed with a heavy meal and before sleeping, the residence time in the colon will be longer, and conversion to enterolactone, the final metabolite, might increase. Enterolignans appear in plasma 8–10 h after consumption. Thus, increased conversion during the night might explain the high concentrations of enterolactone in the morning.

Quantitatively, enterolactone was the major metabolite of the plant lignans in flaxseed. The percentage of enterolactone differed slightly among the supplement periods. When subjects consumed ground flax, the percentage of enterolactone was lower than after consumption of whole or crushed flax (Fig. 2). Secoisolariciresinol, the major lignan in flaxseed, is first converted to enterodiol and thereafter to enterolactone by intestinal bacteria (34). The capacity of the bacteria to convert enterodiol to enterolactone might be limiting when the highly accessible secoisolariciresinol diglucoside of ground flaxseed is given. This is supported by 2 studies (3,35) in which the investigators observed decreasing percentages of enterolactone in urine with increasing consumption of ground flaxseed.

Overall enterolignan concentrations at the end of the 3 wash-out periods did not increase during this study. In addition, there was no effect of treatment order. This suggests that the bacteria in our 9-wk study were not stimulated and that we measured only the effect of milling and crushing of flax on lignan bioavailability. Although we found no evidence for it, stimulation of the colon bacteria due to chronic supplementation with flaxseed was suggested to occur in studies with longer exposures. In rats, Nicolle et al. (36) observed that 2 wk after supplementation with flaxseed, urinary enterolactone concentrations were still increasing. Similarly, in humans, plasma enterolactone concentrations further increased after 2 mo supplementation with flaxseed (3,11).

Throughout this study, the subjects consumed a diet poor in lignans. Although no dietary information was recorded, the low baseline concentrations suggest that compliance with the diet was good. However, in one subject, enterolignan concentrations were high (range 77–191 nmol/L) at the end of the first run-in/wash-out period. This was probably due to dietary noncompliance although the subject did not report this. In the data analysis, we excluded this value because at the end of the other run-in/wash-out periods, the concentrations of both enterolignans were much lower in this subject and comparable to the baseline concentrations of other participants. We expected that this high enterolignan concentration would not affect the next sampling point because the length of the run-in/wash-out period was 11 d. When we excluded all outcomes of this subject, the relative bioavailability changed <3%.

In conclusion, this study shows that bacteria in the colon are able to convert lignans from whole flaxseed to enterolignans, but crushing and milling of flaxseed substantially enhances the bioavailability of lignans from flax. The availability of lignans from food products, such as breads, will improve when whole seeds are replaced by crushed or ground seeds. The food matrix may thus be an important determinant of the bioavailability of lignans. Therefore, when assessing dietary exposure to enterolignans, it is important to determine the bioavailability of lignans from other food sources, especially seeds. Enterolactone was the major metabolite in our study. Yet, the percentage of enterolignans was quite variable among individuals. Even within individuals the percentage of enterolignans varied slightly; the percentage of enterolactone was lower when subjects consumed ground flaxseed instead of whole flaxseed. Plasma enterolactone and enterodiol are expected to be suitable biomarkers because distinction between individuals is feasible as indicated by relatively high ICCs.

ACKNOWLEDGMENTS

The authors thank Michel Buijsman for his excellent technical assistance and Lucy Okma for blood sampling.

FOOTNOTES

¹ Supported by the Netherlands Organization for Health Research and Development (Program Nutrition: Health, Safety, and Sustainability, Grant 014-12-014) and by the Dutch Ministry of Agriculture, Nature and Food Quality.

Manuscript received 24 June 2005. Initial review completed 26 August 2005. Revision accepted 16 September 2005.

LITERATURE CITED

1. Thompson L. U., Robb P., Serraino M., Cheung F. Mammalian lignan production from various foods. Nutr. Cancer. 1991;16:43-52.[Medline]

2. Heinonen S., Nurmi T., Liukkonen K., Poutanen K., Wähälä K., Deyama T., Nishibe S., Adlercreutz H. In vitro metabolism of plant lignans: New precursors of mammalian lignans enterolactone and enterodiol. J. Agric. Food Chem. 2001;49:3178-3186.[Medline]

3. Nesbitt P. D., Lam Y., Thompson L. U. Human metabolism of mammalian lignan precursors in raw and processed flaxseed. Am. J. Clin. Nutr. 1999;69:549-555.[Abstract/Free Full Text]

4. Mazur W. M., Uehara M., Wähälä K., Adlercreutz H. Phyto-oestrogen content of berries, and plasma concentrations and urinary excretion of enterolactone after a single strawberry-meal in human subjects. Br. J. Nutr. 2000;83:381-387.[Medline]

5. Kuijsten A., Arts I.C.W., Vree T. B., Hollman P.C.H. Pharmacokinetics of enterolignans in healthy men and women consuming a single dose of secoisolariciresinol diglucoside. J. Nutr. 2005;135:795-801.[Abstract/Free Full Text]

6. Kirkman L. M., Lampe J. W., Campbell D. R., Martini M. C., Slavin J. L. Urinary lignan and isoflavonoid excretion in men and women consuming vegetable and soy diets. Nutr. Cancer. 1995;24:1-12.[Medline]

7. Kurzer M. S., Lampe J. W., Martini M. C., Adlercreutz H. Fecal Lignan and isoflavonoid excretion in premenopausal women consuming flaxseed powder. Cancer Epidemiol. Biomark. Prev. 1995;4:353-358.[Abstract]

8. Juntunen K. S., Mazur W. M., Liukkonen K. H., Uehara M., Poutanen K. S., Adlercreutz H.C.T., Mykkanen H. M. Consumption of wholemeal rye bread increases serum concentrations and urinary excretion of enterolactone compared with consumption of white wheat bread in healthy Finnish men and women. Br. J. Nutr. 2000;84:839-846.[Medline]

9. Hutchins A. M., Martini M. C., Olson B. A., Thomas W., Slavin J. L. Flaxseed influences urinary lignan excretion in a dose-dependent manner in postmenopausal women. Cancer Epidemiol. Biomark. Prev. 2000;9:1113-1118.[Abstract/Free Full Text]

10. Hutchins A. M., Lampe J. W., Martini M. C., Campbell D. R., Slavin J. L. Vegetables, fruits, and legumes: effect on urinary isoflavonoid phytoestrogen and lignan excretion. J. Am. Diet. Assoc. 1995;95:769-774.[Medline]

11. Tarpila S., Aro A., Salminen I., Tarpila A., Kleemola P., Akkila J., Adlercreutz H. The effect of flaxseed supplementation in processed foods on serum fatty acids and enterolactone. Eur. J. Clin. Nutr. 2002;56:157-165.[Medline]

12. Stumpf K., Pietinen P., Puska P., Adlercreutz H. Changes in serum enterolactone, genistein, and daidzein in a dietary intervention study in Finland. Cancer Epidemiol. Biomark. Prev. 2000;9:1369-1372.[Abstract/Free Full Text]

13. Vanharanta M., Voutilainen S., Lakka T. A., van der Lee M., Adlercreutz H., Salonen J. T. Risk of acute coronary events according to serum concentrations of enterolactone: a prospective population-based case-control study. Lancet. 1999;354:2112-2115.[Medline]

14. Vanharanta M., Voutilainen S., Rissanen T. H., Adlercreutz H., Salonen J. T. Risk of cardiovascular disease-related and all-cause death according to serum concentrations of enterolactone: Kuopio Ischaemic Heart Disease Risk Factor Study. Arch. Intern. Med. 2003;163:1099-1104.[Abstract/Free Full Text]

15. Dai Q., Franke A. A., Jin F., Shu X. O., Hebert J. R., Custer L. J., Cheng J. R., Gan Y. T., Zheng W. Urinary excretion of phytoestrogens and risk of breast cancer among Chinese women in Shanghai. Cancer Epidemiol. Biomark. Prev. 2002;11:815-821.[Abstract/Free Full Text]

16. Ingram D., Sanders K., Kolybaba M., Lopez D. Case-control study of phyto-oestrogens and breast cancer. Lancet. 1997;350:990-994.[Medline]

17. Pietinen P., Stumpf K., Mannisto S., Kataja V., Uusitupa M., Adlercreutz H. Serum enterolactone and risk of breast cancer: a case-control study in eastern Finland. Cancer Epidemiol. Biomark. Prev. 2001;10:339-344.[Abstract/Free Full Text]

18. Olsen A., Knudsen K.E.B., Thomsen B. L., Loft S., Stripp C., Overvad K., Moller S., Tjonneland A. Plasma enterolactone and breast cancer incidence by estrogen receptor status. Cancer Epidemiol. Biomark. Prev. 2004;13:2084-2089.[Abstract/Free Full Text]

19. Grace P. B., Taylor J. I., Low Y. L., Luben R. N., Mulligan A. A., Botting N. P., Dowsett M., Welch A. A., Khaw K. T., Wareham N. J., Day N. E., Bingham S. A. Phytoestrogen concentrations in serum and spot urine as biomarkers for dietary phytoestrogen intake and their relation to breast cancer risk in European Prospective Investigation of Cancer and Nutrition-Norfolk. Cancer Epidemiol. Biomark. Prev. 2004;13:698-708.[Abstract/Free Full Text]

20. Kilkkinen A., Virtamo J., Vartiainen E., Sankila R., Virtanen M. J., Adlercreutz H., Pietinen P. Serum enterolactone concentration is not associated with breast cancer risk in a nested case-control study. Int. J. Cancer. 2004;108:277-280.[Medline]

21. Zeleniuch-Jacquotte A., Adlercreutz H., Shore R. E., Koenig K. L., Kato I., Arslan A. A., Toniolo P. Circulating enterolactone and risk of breast cancer: a prospective study in New York. Br. J. Cancer. 2004;91:99-105.[Medline]

22. Hulten K., Winkvist A., Lenner P., Johansson R., Adlercreutz H., Hallmans G. An incident case-referent study on plasma enterolactone and breast cancer risk. Eur. J. Nutr. 2002;41:168-176.[Medline]

23. den Tonkelaar I., Keinan-Boker L., Van’t Veer P., Arts C.J.M., Adlercreutz H., Thijssen J.H.H., Peeters P.H.M. Urinary phytoestrogens and postmenopausal breast cancer risk. Cancer Epidemiol. Biomark. Prev. 2001;10:223-228.[Abstract/Free Full Text]

24. Milder I.E.J., Arts I.C.W., Putte B., Venema D. P., Hollman P.C.H. Lignan contents of Dutch plant foods: a database including lariciresinol, pinoresinol, secoisolariciresinol and matairesinol. Br. J. Nutr. 2005;93:393-402.[Medline]

25. Milder I.E.J., Feskens E. J., Arts I.C.W., de Mesquita H. B., Hollman P.C.H., Kromhout D. Intake of the plant lignans secoisolariciresinol, matairesinol, lariciresinol, and pinoresinol in dutch men and women. J. Nutr. 2005;135:1202-1207.[Abstract/Free Full Text]

26. Valsta L. M., Kilkkinen A., Mazur W., Nurmi T., Lampi A. M., Ovaskainen M. L., Korhonen T., Adlercreutz H., Pietinen P. Phyto-oestrogen database of foods and average intake in Finland. Br. J. Nutr. 2003;89(suppl. 1):S31-S38.[Medline]

27. Strom S. S., Yamamura Y., Duphorne C. M., Spitz M. R., Babaian R. J., Pillow P. C., Hursting S. D. Phytoestrogen intake and prostate cancer: a case-control study using a new database. Nutr. Cancer. 1999;33:20-25.[Medline]

28. Milder I.E.J, Arts I.C.W., Venema D. P., Lasaroms J. J., Wähälä K., Hollman P.C.H. Optimization of a liquid chromatography-tandem mass spectrometry method for quantification of the plant lignans secoisolariciresinol, matairesinol, lariciresinol, and pinoresinol in foods. J. Agric. Food Chem. 2004;52:4643-4651.[Medline]

29. Kuijsten A., Buijsman M.N.C.P., Arts I.C.W., Mulder P. P., Hollman P.C.H. A validated method for the quantification of enterodiol and enterolactone in plasma using isotope dilution liquid chromatography with tandem mass spectrometry. J. Chromatogr. B. 2005;822:178-184.

30. Rowland M., Tozer T. N. A validated method for the quantification of enterodiol and enterolactone in plasma using isotope dilution liquid chromatography with tandem mass spectrometry. Clinical Pharmacokinetics: Concepts and Application. 3rd ed. Williams and Wilkins Baltimore, MD.

31. Hausner H., Johnsen N. F., Hallund J., Tetens I. A single measurement is inadequate to estimate enterolactone levels in Danish postmenopausal women due to large intraindividual variation. J. Nutr. 2004;134:1197-1200.[Abstract/Free Full Text]

32. Stumpf K., Adlercreutz H. Short-term variations in enterolactone in serum, 24-hour urine, and spot urine and relationship with enterolactone concentrations. Clin. Chem. 2003;49:178-181.[Free Full Text]

33. Zeleniuch-Jacquotte A., Adlercreutz H., Akhmedkhanov A., Toniolo P. Reliability of serum measurements of lignans and isoflavonoid phytoestrogens over a two-year period. Cancer Epidemiol. Biomark. Prev. 1998;7:885-889.[Abstract]

34. Wang L. Q., Meselhy M. R., Li Y., Qin G. W., Hattori M. Human intestinal bacteria capable of transforming secoisolariciresinol diglucoside to mammalian lignans, enterodiol and enterolactone. Chem. Pharm. Bull. 2000;48:1606-1610.[Medline]

35. Lampe J. W., Martini M. C., Kurzer M. S., Adlercreutz H., Slavin J. L. Urinary lignan and isoflavonoid excretion in premenopausal women consuming flaxseed powder. Am. J. Clin. Nutr. 1994;60:122-128.[Abstract/Free Full Text]

36. Nicolle C., Manach C., Morand C., Mazur W., Adlercreutz H., Rémésy C., Scalbert A. Mammalian lignan formation in rats fed a wheat bran diet. J. Agric. Food Chem. 2002;50:6222-6226.[Medline]

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