Plasma Alkylresorcinols and Urinary Alkylresorcinol Metabolites As Biomarkers of Cereal Fiber Intake in Finnish Women

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Short Communication

Plasma Alkylresorcinols and Urinary Alkylresorcinol Metabolites as Biomarkers of Cereal Fiber Intake in Finnish Women

Myle`ne Aubertin-Leheudre, Anja Koskela, Annukka Marjamaa, and Herman Adlercreutz Folkha¨lsan Research Center, Institute of Preventive Medicine, Nutrition and Cancer, and Division of Clinical Chemistry, University of Helsinki, Helsinki, Finland

Abstract

Alkylresorcinols (AR) could be good biomarkers of with plasma AR C17:0 (r = 0.387), AR C19:0 (r = 0.350), consumption offiber-richcereal products. The aim of AR C21:0 (r = 0.428), AR C23:0 (r = 0.409), AR C25:0 this study was to examine the relationship between (r = 0.283), and total AR (r = 0.406) and with urinary AR plasma ARs or urinary AR metabolites and cereal fiber metabolites DHBA (r = 0.359) and DHPPA (r = 0.402) intake in women consuming their habitual diet. even after adjustment for body mass index and age, Twenty-five postmenopausal and 31 premenopausal which could be confounding variables. This is the first women were recruited. The subjects included also study to show a significant correlation between plasma vegetarians (n = 20) to obtain a broad range ofcereal ARs or urinary AR metabolites and cereal fiber intake intake. Dietary intake, plasma ARs, and urinary AR during consumption ofa habitual diet. These results metabolites [3,5-dihydroxybenzoic acid and 3-(3,5-dihy- indicate that assay ofplasma ARs or urinary AR droxyphenyl)-1-propanoic acid] were measured. Pear- metabolites may be used as biomarkers in epidemio- son’s and Partial correlation tests were done between logic studies in free-living populations to evaluate the dietary fiber intake and plasma ARs or urinary AR role ofcereal fiberintake in various diseases. (Cancer metabolites. Cereal fiber intake correlated significantly Epidemiol Biomarkers Prev 2008;17(9):2244–8)

Introduction

Alkylresorcinols (AR) are a group of phenolic lipids Kingdom and around 40 mg/day in Finland (9, 10), and abundant in the outer fiber layers of rye and wheat that 60% of this amount is absorbed (9). grains and absent in highly refined white flour and in The regular consumption of whole grain foods has most other foods (1, 2). Very small amounts are found in been associated with many positive health effects some other cereals. In grains, they exist mainly with alkyl including a reduction in the risk of cardiovascular chain lengths C15:0-C25:0 (3). It has been proposed that disease, diabetes, obesity, and certain types of cancer ARs could function as biomarkers of human whole grain (11). In fact, whole grains slow the digestion, tend to have intake (1, 4). In human subjects, ARs have been detected a low glycaemic index, and could improve insulin in intact form in plasma (4) and as metabolites [3,5- sensitivity (12). The total mortality of postmenopausal dihydroxybenozoic acid (DHBA) and 3-(3,5-dihydroxy- women was negatively associated with whole grain phenyl)-1-propanoic acid (DHPPA)] in urine (5, 6) and intake and positively with refined grain intake (13). The plasma.1 Alkylresorcinols are stable during food process- mechanisms have not been clarified, but different ing (7). Finland and Denmark have the highest intake of compounds in the dietary fiber complex are believed to ARs compared with other European countries (8). play important roles (14). Studies have reported that the differences in consump- It has been suggested that plasma AR concentration tion of ARs are due to age but not to body mass index could be a useful biomarker of whole grain wheat and rye (BMI) because older people change their dietary habits, intake and an indicator of the bread type consumed (15, increasing the amount of whole grain cereal intake (8). It 16). It has been shown that in rye and wheat cereals, the has been estimated that the average daily intake of ARs quantitatively most important ARs are C19:0 (31-32% in free-living population is around 11 mg/day in United- versus 29-35%) and C21:0 (22-25% versus 46-51%). The amount of AR C17:0 is close to AR C21:0 in rye cereals (2). The AR C17:0/C21:0 ratio seems to be a good indicator of Received 3/10/08; revised 5/28/08; accepted 7/2/08. whether a flour or cereal product contains whole-grain Grant support: Medical Research Council in the Academy of Finland, the Sigrid wheat or rye or a combination of these two cereals (7), and Juse´lius Foundation and Sanfundet Folkha¨lsan. Requests for reprints: Myle`ne Aubertin-Leheudre, Folkha¨lsan Research Center, Biomedicum Helsinki, Haartmaninkatu 8 (P.O. Box 63), FIN-00014 University of Helsinki. Phone: 358-9-191-25455; Fax: 358-9-191 25452. 1 Koskela A, Samaletdin A, Aubertin-Leheudre M, Adlercreutz H, E-mail: [email protected] Quantification of AR Metabolites in Plasma by High-Performance Liquid Copyright D 2008 American Association for Cancer Research. Chromatography with Coulometric Electrode Array Detection-J Agric doi:10.1158/1055-9965.EPI-08-0215 Food Chem 2008, published on web 08/09/2008.

Cancer Epidemiol Biomarkers Prev 2008;17(9). September 2008

this is reflected in the plasma values (15). The AR C17:0/ ethical committee of the Helsinki University Central C21:0 ratio is around 0.1 in wheat and 1.0 in rye (7). Hospital approved the research program (Table 1). In some studies, plasma or urinary enterolactone have Data Collection. Subjects were studied for 5 d on 2 been found to be a relatively good biomarker of cereal occasions (in spring and in autumn). The 5-d food record fiber intake (17), but today, enterolactone is regarded to was initiated 2 d before the 72-h collection of plasma and be a better biomarker of total fiber intake and in general urine. The food records included at least 1-week-end reflects a healthy life-style (17). We may conclude that day. The blood samples were taken every morning there are no accepted markers of whole grain cereal during the urine collection. Thus, plasma and urinary intake but ARs seem to be good candidates for this role. samples were obtained simultaneously. The aim of this study was to examine (a)the relationship between plasma intact ARs and urinary Dietary Fiber Intake. Subjects were instructed to AR metabolites and (b) the relationship between plasma maintain normal diet throughout the dietary record. It ARs or urinary AR metabolites and cereal fiber intake. has been shown that a 3-d dietary record is valid to estimate dietary intakes in adults without cognitive impairments. Dietary analyses were completed by a Materials and Methods nutritionist using the tables of Southgate for the fiber data (18). For some typical Finnish food (rye products), Subjects. Twenty-five postmenopausal and 31 pre- we used the fiber data given by the manufacturers. The menopausal women living in the Helsinki area were vegetable fiber values include also the legume fiber recruited. Subjects with a history of cancer or any major values. diseases, or using drugs such as oral contraceptives, Analysis ofARs and Urinary AR Metabolites hormone replacement therapy, or antibiotics were excluded. The subjects included also vegetarians (n = Plasma Analysis. The heparin plasma samples were 20) to obtain a broad range of cereal intake. The collected from fasting subjects. Sodium azide (0.1%) and vegetarians could be vegans (without animal products; ascorbic acid (0.1%) were added and the samples were n = 1), lacto-vegetarians (which included milk product; frozen at À20jC (4). n = 11) or lacto-ovo-vegetarians (which included milk For plasma AR analyses, 500 AL of plasma were taken. product and eggs in their diet; n = 8). To be included as a As an internal standard, AR C20:0 was added to each regular vegetarian, women must have been on this diet sample, blank, and to standards. Then, 500 AL of ionized for at least 1 y (mean, 14 y). All subjects agreed to water were added to the samples, mixed gently, and consume during the study the diet as before the incubated at +37jC overnight. After the incubation recruitment. Age, weight, height, BMI (kg/m2), age at samples were cooled down to room temperature. For menarche, type of diet, number of children, menopausal extraction, 3 mL of diethyl ether was added to the status, smoking status, and physical activity level were samples and mixed well for 2 min. The organic phase recorded during the screening visit by questionnaire. All was separated from the water phase by freezing in a dry subjects gave their informed consent and were initially ice-ethanol bath. The procedure was repeated thrice. The interviewed by a doctor who explained the study. The combined organic phases were evaporated to dryness

Table 1.Descriptive data

Variables All subjects (n = 56) Vegetarians (n = 20) Omnivores (n = 36) Age (y) 46 F 13 45 F 12 47 F 13 Weight (kg) 60 F 958F 962F 9 BMI (kg/m2) 22.5 F 2.7 21.5 F 2.3 23.1 F 2.8 Age of menarche (y) 13 F 113F 113F 1 Number of children 1.8 F 1.4 1.5 F 1.7 2.3 F 0.9 Menopausal status (%) 43 42 44 Nonphysically active (%) 42 37 46 Nonsmoker (%) 87 89 85 Omnivore diet (%) 64 — 100 Urinary DHBA by HPLC (Amol/24 h) 26.8 F 15.6 32.8 F 21.6 24.2 F 15.6 Urinary DHPPA by HPLC (Amol/24 h) 40.2 F 27.6 54.1 F 39.9 34.4 F 17.8 Plasma AR C17:0 by GC/MS (nmol/L) 10.5 F 5.7 9.2 F 4.3 10.9 F 6.1 Plasma AR C19:0 by GC/MS (nmol/L) 19.3 F 12.2 17.6 F 8.8 20.1 F 13.4 Plasma AR C21:0 by GC/MS (nmol/L) 17.3 F 9.5 16.9 F 8.7 17.5 F 9.8 Plasma AR C23:0 by GC/MS (nmol/L) 11.3 F 6.0 10.3 F 4.6 11.7 F 6.5 Plasma AR C25:0 by GC/MS (nmol/L) 7.1 F 3.9 6.9 F 3.2 7.2 F 4.2 Plasma AR C17:0/21:0 ratio (nmol/L) 0.62 F 0.22 0.59 F 0.18 0.64 F 0.23 Total plasma ARs (C17:0-C25:0; nmol/L) 65.5 F 34.7 60.9 F 26.6 67.3 F 37.5 Cereal fiber intake (g/d) 10.0 F 3.0 11.0 F 3.6 9.5 F 2.6 Berry fiber intake (g/d) 3.41 F 3.80 4.88 F 5.29 2.59 F 2.36 Legume fiber intake (g/d) 0.35 F 0.67 0.62 F 1.08 0.20 F 0.12 Vegetable fiber intake (g/d) 4.14 F 2.26 5.51 F 2.73 3.38 F 1.52 Total fiber intake (g/d) 19.8 F 6.3 24.0 F 7.3 17.5 F 4.2

NOTE: Means F SD or %. Abbreviations: HPLC, high performance liquid chromatography; GC/MS, gas chromatography-mass spectroscopy.

Cancer Epidemiol Biomarkers Prev 2008;17(9). September 2008

and redissolved in 300 AL of methanol and further We observed that all ARs correlated significantly with purified using DEAE-Sephadex ion exchange chroma- cereal fiber intake (Fig. 1). We found only one significant tography (in 0.5 Â 1.5-cm columns) in the free base form correlation between total fiber intake and urinary DHBA (19). After the samples were transferred to the columns, (r = 0.300; P = 0.025). No significant difference was neutral steroids were eluted with 6 mL of methanol and detected between plasma ARs and vegetable or berry/ discarded. ARs were eluted with 6 mL 0.1 mol/L acetic fruit fiber intake. acid in methanol. The AR fraction was evaporated to Cereal fiber intake correlated significantly with plasma dryness and derivatized with 100 AL of silanizing reagent AR C17:0 (r = 0.387; P = 0.004), AR C19:0 (r = 0.350; P = [pyridine:hexamethyldisilazane:trimethychlorsilane 9:3:1 0.010), AR C21:0 (r = 0.428; P = 0.001), AR C23:0 (r = (v:v:v)] for gas chromatography-mass spectroscopy 0.409; P = 0.002), AR C25:0 (r = 0.283; P = 0.040), and total analysis (4). ARs (r = 0.406; P =0.003),andwithurinaryAR metabolites DHBA (r = 0.359; P = 0.008) and DHPPA Urine Analysis. The 72-h urine samples were collected (r = 0.402; P = 0.003; Fig. 1) even after adjustment for BMI in plastic bottles containing 1 g of ascorbic acid per liter and age. volume. During collection, the bottles were stored as cool We observed a significant correlation between total as possible and brought to the laboratory every morning plasma ARs and cereal fiber intake both in vegetarians and stored at +4jC. After the 3-d collection, the 24-h urine (r = 0.548; P = 0.018) and in omnivores (r = 0.372; samples were pooled, volume was measured, and after P = 0.033) even after adjustment for BMI and age. addition of 0.1% of sodium azide to counteract bacterial Finally, a stepwise linear regression analysis was done activity. All samples were stored at À20jC and analyzed with plasma ARs (17, 19, 21, 23, 25, and total), DHBA, for AR metabolites using the published protocol A (5). To DHPPA, BMI, age, and enterolactone in the model. We 100 AL of urine, we added 422 ng of internal standard observed that AR C21:0 and DHPPA were both inde- Syringic Acid in 10 AL of methanol. The sample was pendent predictors of cereal fiber intake, explaining 26% hydrolyzed overnight at 37jC with equal volume of the variance (adjusted r2 = 0. 264; P = 0.001). (100 AL) of hydrolysis solution containing 0.1 mol/L Na-acetate buffer (pH 5), 0.2 U/mL h-glucuronidase, and A 2 U/mL sulfatase. After incubation, 50 L aliquot (equal Discussion to 25 AL of urine) were removed and 50 AL of methanol, A and 650 L high performance liquid chromatography The aim of this study was to examine (a) the relationship mobile phase 20% B/A were added to the sample, and between plasma ARs and urinary AR metabolites and (b) the sample was analyzed for the two AR metabolites, the relationship between plasma ARs or urinary AR DHPPA and DHBA, by high performance liquid chro- metabolites and cereal fiber intake. matography with coulometric electrode array detection We observed that cereal fiber intake during a habitual (ESA Biosciences). diet correlates significantly with all plasma ARs and Statistical Analysis. Normality of distribution was urinary AR metabolites in women even after adjustment determined using the Skewness test. All AR and diet for BMI and age, which could be confounding variables variables were log transformed for statistical analysis. (Fig. 1). Furthermore, our results are not influenced by First, we used Pearson’s correlation test to examine the the diet status of our subjects. This indicates that ARs are relation between urinary AR metabolites and plasma AR good biomarkers of cereal fiber intake. This result is levels and between dietary fiber intake and plasma ARs important because at present, there are no accepted or urinary AR metabolites. We calculated Partial corre- biomarkers of whole grain cereal product intake (20). lations between individual ARs and cereal fiber intake Because lignans are abundant in whole grain products using BMI and age as covariables because they could be and correlations were found with high rye bread intake confounding variables. Furthermore, we calculated par- (21, 22), the main metabolite enterolactone was originally tial correlation between total plasma AR sand cereal fiber suggested to be a biomarker of whole grain intake. intake using BMI and age as covariables based on diet However, it is derived from several dietary sources (23), status (vegetarians or omnivores). Finally, stepwise and in regions with low whole grain intake, no consistent regression analysis was used to determine the indepen- correlation between enterolactone and whole grain dent predictors of cereal fiber intake. P values of V0.05 cereal intake has been reported. Thus, enterolactone were considered statistically significant. Analyses were seems not suitable as a specific biomarker of whole grain done using SPSS 15.0 program. intake (24). Epidemiologic studies suggest that a regular con- Results sumption of whole grain foods is associated with a decreased risk of cardiovascular disease and some types We observed that urinary DHBA correlated significantly of cancer (25, 26). The main problem in epidemiologic with plasma AR C17:0 (r = 0.503), AR C19:0 (r = 0.454), studies is to estimate precisely the intake of various food AR C21:0 (r = 0.432), AR C23:0 (r = 0.442), AR C25:0 (r = components. There are some inherent weaknesses of 0.458), and total AR (r = 0.481). Urinary DHPPA food frequency questionnaires (14, 27). The use of a correlated also significantly with plasma AR C17:0 (r = biomarker, which can be quantified, could confirm and 0.456), AR C19:0 (r = 0.416), AR C21:0 (r = 0.395), AR strengthen the conclusions made in such studies (27). C23:0 (r = 0.422), AR C25:0 (r = 0.467), and total ARs (r = The lack of significant correlation between plasma ARs 0.450). Thus, all plasma ARs correlated significantly with and the other fibers confirmed the specificity of ARs as urinary AR metabolites. Plasma AR C17:0, C19:0 and biomarker for cereal rye and wheat fiber intake. Our C25:0 had the highest Pearson’s correlation coefficients results are also not influenced by age or BMI, which are with urinary DHBA and DHPPA. known as confounding variables (10). Moreover, in

Cancer Epidemiol Biomarkers Prev 2008;17(9). September 2008

Figure 1. Pearson’s correlation between cereal fiber intake and plasma ARs/urinary AR metabolites.

Cancer Epidemiol Biomarkers Prev 2008;17(9). September 2008

previous studies, it has been reported that ARs are 5. Koskela A, Linko-Parvinen AM, Hiisivuori P, et al. Quantification of alkylresorcinol metabolites in urine by HPLC with coulometric present in cereal grains, specifically C19:0 and C21:0 in electrode array detection. Clin Chem 2007;53:1380 – 3. rye, in wheat, in triticale, and in barley, and C17:0 in 6. Ross AB, Aman P, Kamal-Eldin A. Identification of cereal alkylre- rye (2, 7). It has been also shown that rye (average sorcinol metabolites in human urine-potential biomarkers of whole- AR content, 734 Ag/g) and wheat cereals (average AR grain wheat and rye intake. J Chromatogr B Analyt Technol Biomed A Life Sci 2004;809:125 – 30. content, 583 g/g) contain the highest amount of ARs (2). 7. Chen Y, Ross AB, Aman P, Kamal-Eldin A. Alkylresorcinols as We have shown that after a dietary intervention in markers of whole grain wheat and rye in cereal products. J Agric women, the plasma total AR concentration seemed to be Food Chem 2004;52:8242 – 6. a useful biomarker of whole grain cereal intake (15, 16). 8. Lang R, Jebb SA. Who consumes whole grains, and how much? Proc In the present study, the plasma AR C17:0/21:0 ratio is Nutr Soc 2003;62:123 – 7. F 9. Ross AB, Shepherd MJ, Bach Knudsen KE, et al. Absorption of 0.62 0.22 nmol/L, which indicates that women dietary alkylresorcinols in ileal-cannulated pigs and rats. Br J Nutr consumed both rye and wheat whole grain cereals (7). 2003;90:787 – 94. We also found that plasma AR C21:0 had the highest 10. Ross AB, Becker W, Chen Y, Kamal-Eldin A, Aman P. Intake of P alkylresorcinols from wheat and rye in the United Kingdom and correlation with cereal fiber intake (r = 0.428; = 0.001; Sweden. Br J Nutr 2005;94:496 – 9. Fig. 1), which is in agreement with a content of both rye 11. Slavin J. Whole grains and human health. Nutr Res Rev 2004;17: and wheat in the diet (2, 7). The AR C21:0 was found to 99 – 110. be an independent predictor of cereal fiber intake (r = 12. Pereira MA, Jacobs DR, Jr., Pins JJ, et al. Effect of whole grains on P insulin sensitivity in overweight hyperinsulinemic adults. Am J Clin 0.410; = 0.002) in this population. Nutr 2002;75:848 – 55. Our study had several limitations. First, our study has 13. Jacobs DR, Pereira MA, Meyer KA, Kushi LH. Fiber from whole been carried out in Finnish women who are known to grains, but not refined grains, is inversely associated with all-cause consume a high daily amount of cereal fiber and ARs mortality in older women: the Iowa women’s health study. J Am Coll Nutr 2000;19:326 – 30S. (10). Moreover, the coefficients of correlation are signif- 14. Slavin J. Why whole grains are protective: biological mechanisms. icant but the r values are clinically moderate (r < 0.750). Proc Nutr Soc 2003;62:129 – 34. Hence, further research, in larger free-living populations 15. Linko AM, Juntunen KS, Mykkanen HM, Adlercreutz H. Whole- in other countries is needed to be able to generalize and grain rye bread consumption by women correlates with plasma alkylresorcinols and increases their concentration compared with confirm our findings. low-fiber wheat bread. J Nutr 2005;135:580 – 3. In conclusion, this is the first study to show a 16. Linko-Parvinen AM, Landberg R, Tikkanen MJ, Adlercreutz H, significant correlation between plasma ARs and urinary Penalvo JL. Alkylresorcinols from whole-grain wheat and rye are AR metabolites. This result is important because it transported in human plasma lipoproteins. J Nutr 2007;137:1137 – 42. 17. Adlercreutz H. Phyto-oestrogens and cancer. Lancet Oncol 2002;3: confirmed that urinary AR metabolites are derived from 364 – 73. plasma ARs as suggested by Ross et al. (6). It also 18. Paul AA, Southgate DA. McCance and Widdowson’s ’The composi- indicates that the urinary AR metabolites (5) may be used tion of foods’: dietary fibre in egg, meat and fish dishes. J Hum Nutr as biomarkers in epidemiologic studies on cereal fiber 1979;33:335 – 6. 19. Fotsis T, Adlercreutz H. The multicomponent analysis of estrogens in intake and disease in free-living populations. urine by ion exchange chromatography and GC-MS-I. Quantitation of estrogens after initial hydrolysis of conjugates. J Steroid Biochem 1987;28:203 – 13. Disclosure of Potential Conflicts of Interest 20. Slavin JL, Jacobs D, Marquart L, Wiemer K. The role of whole grains No potential conflicts of interest were disclosed. in disease prevention. J Am Diet Assoc 2001;101:780 – 5. 21. Pietinen P, Stumpf K, Mannisto S, Kataja V, Uusitupa M, Adlercreutz H. Serum enterolactone and risk of breast cancer: a case-control Acknowledgments study in eastern Finland. Cancer Epidemiol Biomarkers Prev 2001;10: 339 – 44. The costs of publication of this article were defrayed in part by 22. Vanharanta M, Voutilainen S, Lakka TA, van der Lee M, Adlercreutz the payment of page charges. This article must therefore be H, Salonen JT. Risk of acute coronary events according to serum hereby marked advertisement in accordance with 18 U.S.C. concentrations of enterolactone: a prospective population-based case- Section 1734 solely to indicate this fact. control study. Lancet 1999;354:2112 – 5. 23. Adlercreutz H. Lignans and human health. Crit Rev Clin Lab Sci 2007;44:483 – 525. 24. Johnsen NF, Hausner H, Olsen A, et al. Intake of whole grains and References vegetables determines the plasma enterolactone concentration of 1. Ross AB, Kamal-Eldin A, Aman P. Dietary alkylresorcinols: absorp- Danish women. J Nutr 2004;134:2691 – 7. tion, bioactivities, and possible use as biomarkers of whole-grain 25. La Vecchia C, Chatenoud L, Negri E, Franceschi S. Session: whole wheat- and rye-rich foods. Nutr Rev 2004;62:81 – 95. cereal grains, fibre and human cancer wholegrain cereals and cancer 2. Ross AB, Shepherd MJ, Schupphaus M, et al. Alkylresorcinols in in Italy. Proc Nutr Soc 2003;62:45 – 9. cereals and cereal products. J Agric Food Chem 2003;51:4111 – 8. 26. Koh-Banerjee P, Rimm EB. Whole grain consumption and weight 3. Ross AB, Kamal-Eldin A, Aman P. Gas chromatographic analysis of gain: a review of the epidemiological evidence, potential mechanisms alkylresorcinols in rye (secale cereale L) grains. J Sci Food Agric 2001; and opportunities for future research. Proc Nutr Soc 2003;62:25 – 9. 81:1405 – 11. 27. Kaaks R, Ferrari P, Ciampi A, Plummer M, Riboli E. Uses and 4. Linko AM, Parikka K, Wahala K, Adlercreutz H. Gas chromato- limitations of statistical accounting for random error correlations, in graphic-mass spectrometric method for the determination of alkyl- the validation of dietary questionnaire assessments. Public Health resorcinols in human plasma. Anal Biochem 2002;308:307 – 13. Nutr 2002;5:969 – 76.

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