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Nutrition and Disease

A Lignan Complex Isolated from Flaxseed Does Not Affect Plasma Lipid Concentrations or Antioxidant Capacity in Healthy Postmenopausal Women^1,2

Jesper Hallund^*, Gitte Ravn-Haren, Susanne Bügel^*, Tine Tholstrup^* and Inge Tetens^**

^* Department of Human Nutrition and Centre for Advanced Food Studies, the Royal Veterinary and Agricultural University, Frederiksberg, Denmark; Department of Toxicology and Risk Assessment, the Danish Institute for Food and Veterinary Research, Søborg, Denmark; and ^** Department of Nutrition, the Danish Institute for Food and Veterinary Research, Søborg, Denmark

³ To whom correspondence should be addressed: E-mail: jeha{at}kvl.dk.

ABSTRACT

A lignan complex rich in the plant lignan secoisolariciresinol diglucoside (SDG) was isolated from flaxseed. SDG is metabolized by the colonic microflora to the mammalian lignans enterodiol (END) and enterolactone (ENL), and was hypothesized to reduce plasma lipid concentrations and improve antioxidant capacity. The aim of this study was to investigate the effects of a lignan complex, providing 500 mg/d of SDG, on serum concentration and urinary excretion of ENL, plasma lipids, serum lipoprotein oxidation resistance, and markers of antioxidant capacity. Healthy postmenopausal women (n = 22) completed a randomized, double-blind, placebo-controlled, crossover study. Women consumed daily a low-fat muffin, with or without a lignan complex, for 6 wk, separated by a 6-wk washout period. Serum ENL concentration, urinary ENL excretion, plasma concentrations of total cholesterol (TC), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), triacylglycerol (TAG), serum lipoprotein oxidation lag time, plasma Trolox-equivalent antioxidant capacity (TEAC), and ferric reducing ability of plasma (FRAP) were measured at the beginning and end of each intervention period. ENL concentrations in serum (P < 0.001) and ENL urinary excretion (P < 0.001) were significantly higher after the lignan complex intervention period compared with placebo. Plasma concentrations of TC, LDL-C, HDL-C, TAG, lipoprotein oxidation lag time, TEAC and FRAP were not affected. Daily consumption for 6 wk of a low-fat muffin enriched with a lignan complex significantly increased serum ENL concentrations and urinary ENL excretion in healthy postmenopausal women, but had no effect on plasma lipid concentrations, serum lipoprotein oxidation resistance, or plasma antioxidant capacity.

KEY WORDS: • humans • secoisolariciresinol diglucoside • enterolactone • phytoestrogens

Cardiovascular disease (CVD)⁴ is the leading cause of death worldwide (1). After menopause, women have an increased risk of CVD due to a more atherogenic lipid profile (2), which has been attributed to a decline in the circulating levels of estrogen (3).

Flaxseed is the richest dietary source of the plant lignan secoisolariciresinol diglucoside (SDG), which is metabolized to the mammalian lignans enterodiol (END) and enterolactone (ENL) by colonic bacteria (4,5). Plant lignans belong to the group of phytoestrogens, which are structurally similar to endogenous estrogen and have binding affinity to the sex steroid-binding globulin (6,7).

Results from randomized controlled studies in normal (8) and hyperlipidemic (9,10) subjects showed that intake of 38–50 g flaxseed reduces serum cholesterol. Further results showed that the nonlipid components in partially defatted flaxseed significantly reduce serum total cholesterol (TC), LDL cholesterol (LDL-C), apolipoprotein (apo) B, apo A-I, and increases triacylglycerol (TAG) concentrations in hyperlipidemic subjects (11). These findings suggest that the cholesterol-lowering effect may be limited to the component of protein, fiber, or plant lignans or a combination of the 3. The health benefits of isolated plant lignans from flaxseed were examined recently in animal models. Those studies showed that isolated SDG from flaxseed reduced TC and LDL-C concentrations by 33 and 35% in rabbits fed a high-cholesterol diet (12). In addition, further results showed that a lignan complex isolated from flaxseed reduced TC and LDL-C concentrations by 20 and 14% in rabbits fed a high-cholesterol diet (13). Based on these results, it was hypothesized that plant lignans from flaxseed may have a cholesterol-lowering effect.

SDG and its metabolites, END and ENL, were shown to exert antioxidant activity in different lipid and aqueous in vitro model systems and to be effective in decreasing lipid oxidation in vitro (12–17). Prasad (12) showed a decrease in aortic malondialdehyde (MDA) concentration, a marker of lipid oxidation, by SDG in rabbits fed a high-cholesterol diet (12); a similar decrease in aortic MDA concentration was observed recently in the same model by a flax lignan complex (13). In humans, a dietary intake of 50 g flaxseed for 4 wk did not affect plasma lipid hydroperoxides in healthy young adults (8), whereas low serum ENL concentrations were associated with increased lipid oxidation in a cross-sectional study (18).

No studies have been conducted in humans to determine whether the SDG lignan component of flaxseed is responsible for the reduction in lipid concentrations or whether it possesses antioxidant activity. Therefore, we conducted a randomized, double-blind, placebo-controlled, crossover study to determine whether a lignan complex isolated from flaxseed would affect endothelial function, inflammation markers, plasma lipids, and plasma antioxidant capacity in healthy postmenopausal women. The main objectives of the present study were to determine whether a lignan complex isolated from flaxseed would 1) increase serum ENL concentration and urinary ENL excretion, 2) reduce plasma lipid concentrations, 3) increase serum lipoprotein resistance to oxidation ex vivo, and 4) affect plasma antioxidant capacity.

SUBJECTS AND METHODS

    Subjects. Healthy postmenopausal women were recruited from Copenhagen and the surrounding areas by advertisement in the local media. The women were 61 ± 7 y old (mean ± SD) and postmenopausal (defined as no menstrual period for >24 mo). Before the study, none of the women had used hormone replacement therapy for at least 6 mo, or fatty acid-, isoflavone-, vitamin- or mineral-containing supplements for 2 mo, or antibiotics for 3 mo. None of the women had any history of diabetes, inflammatory diseases, or CVD and they did not use antihypertensive, anti-inflammatory or lipid-lowering drugs on a regular basis. All women were nonsmokers and had a blood pressure (BP) <160/90 mm Hg. Screening blood samples were taken before entry and all subjects had TC <8 mmol/L, TAG <3 mmol/L, hemoglobin >7.0 mmol/L, and fasting glucose <6.5 mmol/L. A total of 23 women were included in the study. One woman withdrew from the study due to the use of antibiotics during the study period; 22 women completed the study according to the protocol.

    Ethical approval. Ethical approval was obtained from the Local Research Ethical Committee of Copenhagen and Frederiksberg (KF 11–047/03). All women received oral and written information about the study before they gave written informed consent.

    Study design. We performed a randomized, double-blind, placebo-controlled, crossover study. The women consumed a low-fat muffin, with or without a lignan complex, for 6 wk, separated by a 6-wk washout period. The women were instructed to consume the muffin at least 1 h after dinner and to keep daily records of muffin consumption and well-being in a study diary. The muffins were identical in all respects other than enrichment with a lignan complex providing 500 mg/d of SDG. The average nutrient content of each muffin (58 g) was as follows: energy 642 kJ; protein 2.6 g; carbohydrate 34.1 g; fat 0.5 g; and fiber 0.7 g. The muffins were produced in a single batch and were frozen at –20°C until use. The lignan complex was added to the dough just before baking. The major components of the lignan complex were as follows: 32.9% SDG, 13.9% cinnamic acids, 11.8% protein, 10.0% 3-hydroxy-3-methyl glutaric acid, 3.5% fat, 3.3% moisture, and 1.0% ash. The women were instructed to avoid any flaxseed consumption during the study period. Compliance was assessed using study diaries, as well as ENL concentrations in serum and urine.

Frozen muffins were handed out to the women every 2nd wk from the department for consumption at home. In addition, the women visited the department for 5 examinations during the study: 1 wk before the beginning of the study (wk –1); at the beginning of the study (wk 0); after the 1st intervention period (wk 6); at the beginning of the 2nd intervention period after the 6-wk washout period (wk 12); and after the 2nd intervention period (wk 18). Height was measured at wk 0. Body weight, BP, 24-h urinary ENL excretion, and plasma concentrations of TC, TAG, HDL cholesterol (HDL-C), serum lipoprotein oxidation lag time, Trolox-equivalent antioxidant capacity (TEAC), and ferric reducing ability of plasma (FRAP) were measured at wk 0, 6, 12, and 18. A 3-d weighed food record was completed 1 wk before the study and during the last week of each intervention period to estimate habitual dietary intake. Energy and nutrient intake were calculated using the Dankost 2000 dietary assessment software (National Food Agency).

Blood samples were collected from subjects during the morning after they had fasted for 12 h and had rested for 15 min in a supine position. A total of 180 mL blood was collected during the entire study. Women were instructed to consume a standardized low fat meal providing a maximum of 15 g fat the evening prior to blood collection.

    Laboratory measurements. For the analysis of serum ENL, blood samples were drawn from fasting subjects into 5-mL tubes with no additives (Becton Dickinson 366434), centrifuged at 3000 x g for 15 min at 20°C, and stored at –20°C until further analysis. Urinary excretion over 24 h was colleted into 2.5-L containers with 2 g added boric acid and stored at –20°C until further analysis. Serum and urine ENL concentrations were determined using time-resolved fluoroimmunoassay (Labmaster Diagnostics) as previously described (19–21). The intra- and interassay CV were 15.6 and 14.0%, and 10.2 and 8.3% for serum and urinary ENL, respectively.

Blood samples were drawn from fasting subjects into 5-mL EDTA tubes (Becton Dickinson 366457) for the analysis of TC, HDL-C, and TAG, centrifuged at 1600 x g for 10 min at 4°C, and stored at –20°C until further analysis. A Cobas Mira⁺ analyzer (Roche Diagnostic) was used to measure TC (CHOD-PAP, Roche Diagnostic) and HDL-C (HDL-C-plus 2nd generation, Roche Diagnostic) and TAG (GPO-PAP, Roche Diagnostic) by enzymatic kits. LDL-C concentrations were calculated using the Friedewald formula (22). The intra- and interassay CV were 0.9 and 1.5%, 1.8 and 3.5%, and 0.6 and 3.3% for TC, HDL-C, and TAG, respectively.

Blood samples were drawn from fasting subjects into 10-mL tubes with no additives (Becton Dickinson 366434) for the analysis of serum lipoprotein oxidation lag time, centrifuged at 1600 x g for 10 min at 4°C, and stored at –80°C until further analysis. Serum lipoprotein oxidation lag time was determined according to Mayer et al. (23). The time-dependent decrease in fluorescence intensity was followed using a Wallac 1420 multilabel counter (PerkinElmer Danmark A/S). The fluorescent marker diphenylhexatriene-labeled phosphatidylcholine (DPHPC) was purchased from Chemica Technologies. The interday CV in lagtime for a control serum sample was 4.3%.

Blood samples were drawn from fasting subjects into 5-mL EDTA tubes (Becton Dickinson 366457) for the analysis of TEAC and FRAP, centrifuged at 1600 x g for 10 min at 4°C, and stored at –20°C until further analysis. TEAC (24) was determined using a commercially available kit (NX2332, Randox Laboratories) and FRAP as previously described (25). Intraday CV in TEAC and FRAP of the control sample were 4.3 and 0.9%, respectively.

    Power calculations. The number of women needed in this study was calculated using the method of least standardized difference (26). The study was designed primarily to demonstrate a difference in markers of endothelial function. The inclusion of 23 women gave the study enough power (80%) to detect a significant difference (P < 0.05) of 0.65 x SD of the study outcome measures, which equals a 10% difference in TC.

    Statistical analysis. Data describing the characteristics of the volunteers are summarized as means ± SD. Data on the outcome of the study are expressed as means ± SEM. Data were analyzed in SAS 8.02 (© SAS Institute) using a mixed model analysis of covariance with treatment (lignan complex muffin or placebo muffin) and period (first or second period) as fixed factors, subjects as random factor, and baseline measurements as a covariate. Further fixed terms corresponding to treatment/period interactions were included to test for any carry-over effect between periods, and the treatment/covariate interaction was included to test whether the treatment effect of the lignan complex varied according to the baseline values of the covariate. For TC, LDL-C, and HDL-C, the data were logarithmically transformed to obtain normally distributed residuals. Differences in habitual energy and nutrient intake calculated 1 wk before the study and during the last week of each intervention period were analyzed using ANOVA.

RESULTS

    Baseline characteristics and compliance. The baseline characteristics of the women were as follows: age, 61 ± 7 y; BMI, 24.1 ± 3.4 kg/m²; TC, 5.97 ± 1.02 mmol/L; LDL-C, 3.77 ± 1.01 mmol/L; HDL-C, 1.75 ± 0.45 mmol/L; TAG, 0.98 ± 0.26 mmol/L; systolic BP, 124 ± 13 mm Hg; and diastolic BP, 75 ± 8 mm Hg.

The women perceived the lignan complex and placebo muffins to be identical in appearance and taste. Gastrointestinal symptoms were reported by 4 women during the intervention period, and another 4 women reported gastrointestinal symptoms during the placebo period. Headaches were reported by 2 women during the intervention period and by 1 woman during the placebo period. Compliance assessed using study diaries showed that 98% of all muffins were consumed during the 2 intervention periods.

    Body weight and blood pressure. Body weight (P = 0.482), BMI (P = 0.438), systolic BP (P = 0.799), and diastolic BP (P = 0.642) did not differ after the intervention study. Body weight was 67.2 ± 2.0 kg at baseline and 67.6 ± 2.0 kg at the end of the lignan complex treatment compared with 67.8 ± 2.1 kg at baseline and 67.9 ± 2.0 kg at the end of the placebo treatment. BMI was 24.2 ± 0.7 kg/m² at baseline and 24.3 ± 0.7 kg/m² at the end of the lignan complex treatment compared with 24.4 ± 0.7 kg/m² at baseline and 24.4 ± 0.7 kg/m² at the end of the placebo treatment. BP was 124 ± 3/75 ± 2 mm Hg at baseline and 122 ± 3/73 ± 2 mm Hg at end of the lignan complex treatment compared with 123 ± 3/75 ± 2 mm Hg at baseline and 122 ± 3/74 ± 1 mm Hg at end of the placebo treatment.

    Dietary intake. Energy, fat, saturated fatty acid, monounsaturated fatty acid, PUFA, protein, carbohydrate, total fiber, dietary cholesterol, and alcohol intakes calculated at baseline and wk 6 of the intervention and placebo periods did not differ (Table 1).

DISCUSSION

In this double-blind, placebo-controlled, randomized, crossover study, we showed that 6 wk of consumption of a muffin enriched with a lignan complex isolated from flaxseed, providing 500 mg/d SDG, significantly increased ENL concentrations in serum and ENL urinary excretion in healthy postmenopausal women, but had no effect on plasma TC, LDL-C, HDL-C, TAG concentrations, serum lipoprotein oxidation resistance, TEAC, and FRAP.

In the present study, a lignan complex isolated from flaxseed was chosen to focus on the isolated effect of the plant lignans without the physiological effects of fiber, PUFA, and protein. The lignan complex was baked into a low-fat muffin and provided to the women in 1 daily dose. Earlier studies showed that serum ENL concentrations can be maintained with 1 daily dose of lignans after a period of constantly high lignan intake (27). In addition, SDG were shown to be stable to the baking process and almost all SDG is recovered (28). The dose of 500 mg/d of SDG used in our study corresponds to 21–42 g defatted flaxseed or 38–82 g whole flaxseed according to recent analysis of SDG content in flaxseed (29). The dose was chosen to allow for comparison between results in the present study and earlier studies using 38–50 g of ground and defatted flaxseed (8–11).

No other studies have investigated the effect of plant lignans on plasma lipid concentration or serum lipoprotein oxidation resistance and antioxidant capacity in humans. A single study examined the effect of 3 wk supplementation of 50 g of partially defatted flaxseed on lipid concentrations; TC and LDL-C were significantly reduced and HDL-C concentrations increased among hyperlipidemic men and women (11). In that study, canola oil and wheat bran were added to the placebo muffin and compared with a muffin enriched with partially defatted flaxseed to determine whether the nonlipid components, especially the viscous fiber seed coat rich in plant lignans, were responsible for the cholesterol-lowering effect. The authors concluded that the flaxseed gum was likely the major active ingredient in flaxseed responsible for the effect on plasma lipids, but that the flaxseed protein and the flaxseed lignans may have contributed to the observed cholesterol reduction (11). However, more recent animal studies suggested that the plant lignan SDG may be responsible for the cholesterol-lowering effect (12,13). These studies showed that SDG isolated from flaxseed (12) and a lignan complex from flaxseed rich in SDG (13) significantly reduced TC and LDL-C, increased HDL-C in hypercholesterolemic rabbits, and reduced the development of atherosclerotic plaque formations. The doses used in the 2 studies were 40 mg lignan complex/kg body weight (13) and 15 mg SDG/kg body weight (12) for a duration of 8 wk compared with 22.7 mg lignan complex/kg body weight used in our study, providing 7.5 mg SDG/kg body weight.

In our study, plasma lipid concentrations did not differ between the lignan complex and placebo intervention periods. The cholesterol-lowering effects of whole and defatted flaxseed occurred mainly among hyperlipidemic postmenopausal women and adult men (9–11) even though a single study reported a reduction among young healthy adults (8). The women in our study were apparently healthy postmenopausal women with an initial TC concentration of 5.97 ± 1.02 mmol/L. Although these findings cannot be generalized to other phytoestrogens, it may be interesting to examine whether a different treatment effect of the lignan complex would have occurred in hyperlipidemic women with a different CVD risk profile.

It is possible that SDG in flaxseed is not responsible for the effect on plasma lipids. The cholesterol-lowering effect reported in studies examining the effect of whole or defatted flaxseed on plasma lipids may be an effect of the protein or fiber fraction alone or that fraction in combination with SDG. Flaxseed contains 8% viscous polysaccharides (30), which were shown to reduce TC and LDL-C concentrations (31). Protein may also be responsible for the cholesterol-lowering effect in combination with SDG. Soy proteins containing isoflavones were shown to decrease TC, LDL-C, and TAG concentrations in humans (32), whereas a large number of clinical studies conducted among postmenopausal women reported that cholesterol and triglyceride concentrations were unaffected after supplementation with isolated isoflavones without soy protein (33–37).

We found no effect of a lignan complex, providing 500 mg/d of SDG on serum lipoprotein resistance to oxidation, TEAC, and FRAP. It was reported previously that low serum ENL concentrations are associated with increased lipid oxidation in men, determined as plasma F₂-isoprostane concentration (11). The authors found that the strongest predictor of serum ENL concentrations was intake of soluble fibers, which are found in many fruits and vegetables. Resistance of lipoprotein to oxidation was reported previously to increase with intake of fruit and vegetables (38). It is therefore likely that the protective effect on lipid oxidation seen by Vanharanta et al. (18) is due to other associated compounds. Two studies by Prasad investigated the effect of isolated SDG and a lignan complex isolated from flaxseed on lipid oxidation in rabbits (12,13). They found reduced serum MDA concentrations after lignan complex supplementation and reduced aortic MDA concentrations after intake of isolated SDG or lignan complex in rabbits fed a high-cholesterol diet (0.5–1% cholesterol). Isolated SDG or lignan complex had no effect on aortic MDA concentrations in rabbits fed a control diet. These data suggest that a possible beneficial effect of SDG was found only in those rabbits fed a high-cholesterol diet. Our study is the first to test the effect of a lignan complex on resistance to oxidation of serum lipoprotein in humans. Despite the antioxidant activity of SDG measured in vitro, our results do not indicate a protective effect of 6 wk of consumption of a lignan complex, providing 500 mg/d of SDG, on susceptibility of serum lipoprotein to oxidation ex vivo. It is possible that beneficial effects appear only in individuals with elevated oxidative stress, which might explain our results.

The SDG content of flaxseed was not measured in any of the randomly controlled studies investigating the effect of whole or partially defatted flaxseed in humans, which makes comparison difficult. However, the amount of SDG used in our study corresponds to 21–42 g defatted flaxseed or 38–82 g whole flaxseed according to recent analysis of SDG content in flaxseed (29). Serum concentration of ENL reached 385 ± 67 nmol/L, and 24 h urinary ENL excretion was 94 ± 11 µmmol/dafter the 6-wk lignan complex intervention period. No other intervention studies measured serum ENL concentration after flaxseed supplementation, but urinary ENL excretion between 13 and 41 µmmol/d was measured after 25 g ground flaxseed supplementation for 1–16 wk (9,27). In addition, Nesbitt et al. showed that there is a linear dose-response relation between urinary ENL excretion and ingestion of increasing amounts of flaxseed (25 g) (27). The high serum ENL concentration and urinary ENL excretion found in our study suggest that SDG is highly available to the colonic microflora and that it was metabolized to ENL.

In conclusion, 6 wk of consumption of a low-fat muffin enriched with a lignan complex, providing 500 mg/d of SDG, significantly increased serum ENL concentrations and urinary ENL excretion in healthy postmenopausal women, but had no effect on plasma lipid concentrations, serum lipoprotein oxidation resistance, or plasma antioxidant capacity. These results do not suggest that plant lignans isolated from flaxseed would affect plasma lipid concentration or antioxidant capacity in healthy postmenopausal women.

ACKNOWLEDGMENTS

We thank Vibeke Kegel, Leif S. Jakobsen, Pia L. Madsen, Yvonne Rasmussen, Berit Hoielt, and Hanne Jensen for excellent technical support.

FOOTNOTES

¹ Presented in poster form at the 2nd Conference on Polyphenols and Health, 4–7 October 2005, Davis, CA. (Hallund J, Ravn-Haren G, Bügel S, Tholstrup T, Tetens I. A lignan complex isolated from flaxseed increases serum enterolactone concentrations but does not affect plasma lipid concentrations, serum lipoprotein oxidation resistance or plasma antioxidant capacity in healthy postmenopausal women).

² Supported by Ministry of Science, Technology and Innovation (Copenhagen, Denmark), the Commission of the European Communities, ISOHEART QLK1-2001-00221, and by Danish grants from Aase and Ejner Danielsens Foundation and Beckett-foundation. Archer Daniels Midland Company (Decatur, IL) funded the lipid oxidation analysis and provided the lignan complex.

⁴ Abbreviations used: apo, apolipoprotein; BP, blood pressure; CVD, cardiovascular disease; END, enterodiol; ENL, enterolactone; FRAP, ferric reducing ability of plasma; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; MDA, malondialdehyde; SDG, secoisolariciresinol diglucoside; TAG, triacylglycerol; TC, total cholesterol; TEAC, Trolox-equivalent antioxidant capacity.

Manuscript received 12 September 2005. Initial review completed 20 October 2005. Revision accepted 31 October 2005.

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