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The effect of a lignan complex isolated from flaxseed on inflammation markers in healthy postmenopausal women
Nutrition, Metabolism & Cardiovascular Diseases (2008) 18, 497e502
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/nmcd
The effect of a lignan complex isolated from flaxseed on inflammation markers in healthy postmenopausal women ¨gel a, T. Tholstrup a, J.M. Bruun c J. Hallund a,*, I. Tetens b, S. Bu a
Department of Human Nutrition, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg C, Denmark b National Food Institute, Technical University of Denmark, Søborg, Denmark c Department of Endocrinology and Metabolism C, Aarhus University Hospital, Aarhus C, Denmark Received 13 November 2006; received in revised form 30 April 2007; accepted 2 May 2007
KEYWORDS Phyto-oestrogens; Lignans; Flaxseed; Enterolactone; Inflammation
Abstract Background and aim: Plant lignans are metabolised by the colonic micro-flora to the mammalian lignans enterodiol and enterolactone, which are hypothesized to be cardioprotective. The aim of this study was to investigate the effects of a plant lignan complex isolated from flaxseed, providing 500 mg/d of secoisolariciresinol diglucoside, on inflammatory markers. Methods and results: Healthy postmenopausal women (n Z 22) completed a randomised double-blind, placebo-controlled crossover study. Women consumed daily a low-fat muffin, with or without a lignan complex, for 6 weeks, separated by a 6-week washout period. A significant difference of approximately 15% (P Z 0.028) was observed for C-reactive protein (CRP) concentration between the lignan complex intervention period and placebo period. CRP concentrations (median; 25th, 75th percentiles) were 0.88 (0.63, 2.05) mg/L at baseline and 0.92 (0.59, 1.49) mg/L after the lignan complex intervention period compared with 0.80 (0.62, 1.62) mg/L at baseline and 1.10 (0.72, 1.62) mg/L after placebo. No significant differences in interleukin-6, tumor necrosis factor-a, soluble intracellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, and monocyte chemoattractant protein-1 were found between the lignan complex intervention period and placebo period. Conclusion: Daily consumption for 6 week of a low-fat muffin enriched with a lignan complex may reduce CRP concentrations compared to a low-fat muffin with no lignans added. ª 2007 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: þ45 3528 2485; fax: þ45 3528 2483. E-mail address: [email protected] (J. Hallund). 0939-4753/$ - see front matter ª 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.numecd.2007.05.007
498
Introduction Flaxseed is the richest dietary source of the plant lignan secoisolariciresinol diglucoside (SDG), which is metabolised to the mammalian lignans enterodiol and enterolactone (ENL) by colonic bacteria [1]. Plant lignans belong to the group of phyto-oestrogens, which have chemical structures resembling those of endogenous oestrogen and which have binding affinity to the sex steroid-binding globulin [2]. Recently, a number of observational studies have suggested that mammalian lignans may have a protective effect against the risk of cardiovascular disease (CVD) [3e5]. High serum ENL concentrations have prospectively been associated with a reduced risk of acute cardiovascular events and lower coronary heart disease (CHD) and CVDrelated mortality in healthy men [3,6]. We have earlier shown that daily consumption for 6 week of a low-fat muffin enriched with a lignan complex significantly increases serum ENL concentrations and urinary ENL excretion in healthy postmenopausal women, but have no effect on plasma lipid concentrations, serum lipoprotein oxidation resistance, plasma antioxidant capacity [7] or endothelial function [8]. However, a number of controlled clinical intervention studies have shown that whole and defatted flaxseed lowers serum cholesterol in normal and hyperlipidemic subjects [9e11]. Furthermore, isolated SDG [12] as well as a lignan complex isolated from flaxseed [13] have been shown to reduce total cholesterol (TC), LDL cholesterol (LDL-C), and increases HDL cholesterol (HDL-C) in hypercholesterolemic rabbits, and reduce the development of hypercholesterolemic atherosclerosis by 34e73%. To our knowledge, no studies have so far examined the role of plant or mammalian lignans in the inflammation process. Chronic low-grade inflammation and increased adhesiveness for circulating leukocytes by vascular endothelial cells play a key role in the inflammatory process leading to atherosclerosis and CVD [14]. Increased plasma concentrations of the acute-phase reactant C-reactive protein (CRP) have been shown to be a strong predictor of CVD and CHD in apparently healthy women [15], but the underlying mechanism of this association is not understood. CRP is secreted by several cell-types, but primarily hepatocytes in response to IL-6 as well as other pro-inflammatory proteins [16e18]. Recent data suggest that CRP may also be produced in human coronary artery smooth muscle cells and the atherosclerotic lesion [18,19] indicating CRP as an active player in the development of cardiovascular disease. In vitro studies have shown that CRP induce increased expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) in human umbilical vein and coronary artery endothelial cells [20,21]. These studies suggest that CRP may be involved in the pathogenesis of atherosclerosis or inflammation. The adhesion of leukocytes to the vascular endothelium is mediated by cellular adhesion molecules (CAMs), such as ICAM-1 and VCAM-1, and is probably the first step in the atherosclerotic process. Following adhesion of leukocytes to the vascular endothelium, chemoattracting proteins (chemokines), such as MCP-1, play a key role in the migration of the leukocytes into the arterial intima [22].
J. Hallund et al. To our knowledge, no other studies have been published in relation to the possible role of the plant lignan SDG on central markers related to the inflammation process. The main objectives of this study are to determine whether a lignan complex isolated from flaxseed would affect plasma concentration of CRP, IL-6, TNF-a, sICAM-1, sVCAM-1 and MCP-1 in healthy post-menopausal women.
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 months). Prior to the study none of the women had used hormone replacement therapy in the past six months, or fatty acid, isoflavones, vitamin or mineral containing supplements in the past two months, or antibiotics in the past three months. None of the women had any history of diabetes, inflammatory diseases or CVD and they did not use any antihypertensive, anti-inflammatory or lipid lowering drugs. All women were non-smokers and had a BP of less than 160/90 mmHg. Screening blood samples were taken before entry and all subjects had TC <8 mmol/L, TAG <3 mmol/L, hemoglobin >7 mmol/L, and fasting glucose <6.5 mmol/L. A total of twenty-three women were included in the study. One woman withdrew from the study due to the use of antibiotics during the study period. Twenty-two women completed the study according to the protocol. Ethical approval was obtained from the Local Research Ethical Committee of Copenhagen and Frederiksberg (KF 01-130/02). All women received oral and written information about the study before they gave written informed consent.
Study design We performed a randomised, double blind, placebo controlled, crossover study. The women were instructed to consume their habitual diets throughout the study period but avoid any flaxseed consumption. Besides the habitual diet they consumed a low fat muffin daily, with or without a lignan complex, for 6 weeks, separated by a 6-week washout period. The women were instructed to consume the muffin 1 h after the evening meal 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: energy 642 kJ; protein 2.6 g; carbohydrate 34.1 g; fat 0.5 g and fibre 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. Compliance was assessed using study diaries, as well as ENL concentrations in serum and urine.
Flaxseed lignans and inflammatory markers Frozen muffins were handed out to the women every second week from the department for consumption at home. In addition, the women visited the department for five examinations during the study: one week before the beginning of the study (week 1); at the beginning of the study (week 0); after the first intervention period (week 6); at the beginning of the second intervention period after 6week washout period (week 12); and after the second intervention period (week 18). Height was measured at week 0. Body weight, blood pressure (BP), twenty-four hour urinary ENL excretion, and plasma concentrations of CRP, IL-6, TNF-a, sICAM-1, sVCAM-1 and MCP-1 were measured at week 0, 6, 12 and 18. Twelve hour fasting blood samples were collected during the morning after 15 min of supine rest. 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 A fasting blood sample was drawn into a 5 mL EDTA tube (Becton Dickinson #366457) for the analysis of high sensitive CRP (hsCRP). Thereafter, it was centrifuged at 1600 g for 10 min and stored at 20 C until further analysis. HsCRP was measured with a chemiluminescent immunometric assay (Diagnostic Products Corporation) using an Immulite 1000 analyser (Diagnostic Products Corporation). Intra- and inter-assay coefficient of variance was 3.4% and 3.7%, respectively. A fasting blood sample was drawn into a 10mL Na heparin vacutainer (Becton Dickinson #366480) for the IL-6, TNF-a, sICAM1, sVCAM-1 and MCP-1 analysis. Blood was centrifuged at 1600 g for 10 min at 4 C and plasma was stored at 80 C until analysis. IL-6, TNF-a, sICAM-1, sVCAM-1 and MCP-1 concentrations were determined using human IL-6 (Quantikine HS600, R&D Systems Europe Ltd., Abingdon, UK), TNF-a (Quantikine HSTA00C, R&D Systems Europe Ltd., Abingdon, UK), sICAM-1 (Parameter BBE1B, R&D Systems Europe Ltd., Abingdon, UK), sVCAM-1 (Parameter BBE3, R&D Systems Europe Ltd., Abingdon, UK) and MCP-1 (DuoSet DY297, R&D Systems Europe Ltd., Abingdon, UK) immunoassay kits. Intra- and inter-assay CVs were 5.5% and 7.9%, 3.5% and 6.9%, 6.4% and 5.0%, 8.3% and 9.5% and 8.1% and 9.6% for IL-6, TNF-a, sICAM-1, sVCAM-1 and MCP-1, respectively. Serum and urine ENL concentrations were determined using time-resolved fluoroimmunoassay (Labmaster Diagnostics, Turku, Finland) as previously described [7].
499 as means SEM. Data on the outcome of the study are expressed as the median and 25th, 75th percentiles. Data were analysed in SAS 8.02 (ª SAS Institute Inc., Cary, USA) 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 the baseline measurements for each period 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 were included to test if the treatment effect of the lignan complex varied according to the baseline values of the covariate. All data were logarithmictransformed to obtain normally distributed residuals. Differences were considered significant when P < 0.05.
Results The baseline characteristics of the women were as follows (mean SD): age 61 7 y, BMI 24.1 3.4 kg/m2, TC 5.97 1.02 mmol/L, LDL cholesterol 3.77 1.01 mmol/L, HDL cholesterol 1.75 0.45 mmol/L, TAG 0.98 0.26 mmol/L, systolic BP 124 13 mmHg, and diastolic BP 75 8 mmHg. Compliance assessed using study diaries showed that 98% of all muffins were consumed during the two intervention periods. A significant difference between the lignan complex intervention period and placebo period was observed for plasma CRP concentration (P Z 0.028), which was caused by a higher CRP concentration following the placebo period (Fig. 1). CRP concentrations were 0.88 (0.63, 2.05) mg/L at baseline and 0.92 (0.59, 1.49) mg/L after the lignan complex intervention period compared with 0.80 (0.62, 1.62) mg/L at baseline and 1.10 (0.72, 1.62) mg/L after the placebo period (P Z 0.028) (Table 1). There were no significant differences in plasma IL-6, TNF-a, VCAM-1, ICAM-1, and MCP-1 concentrations after the two intervention periods. ENL concentrations in serum and urinary ENL excretion increased from 46 8 nmol/L and 17 2 mmol/d, respectively, at baseline to 385 67 nmol/L (P < 0.001) and 94 11 mmol/d (P < 0.001) respectively, following the lignan complex intervention period. No changes in serum ENL concentrations or urinary ENL excretion were observed during the placebo intervention period. 6,00 5,00
Power calculations mg/L
4,00
The number of women needed in this study was calculated using the method of least standardized difference. 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 SD of the study outcome measures.
3,00 2,00 1,00 0,00
Statistical analysis Data describing the characteristics of the volunteers are summarized as the means SD. Data on ENL are expressed
Week 1
Week 6
Lignan complex
Week 1
Week 6
Placebo
Figure 1 Individual plasma CRP concentration during the lignan complex and placebo intervention period.
500
J. Hallund et al.
Table 1 Plasma concentrations of circulation markers of inflammation in healthy postmenopausal women at baseline (week 0) and week 6 of the lignan complex intervention and placebo periods (n 22) (median values with their 25th and 75th percentiles) Py
Variable*
Lignan complex Baseline
Week 6
Baseline
Week 6
CRP (mg/L) IL-6 (ng/L) TNF-a (ng/L) SICAM-1 (ng/mL) SVCAM-1 (ng/mL) MCP-1 (ng/L)
0.88 1.28 1.08 204 349 238
0.92 1.14 1.07 212 352 226
0.80 1.16 1.06 205 363 255
1.10 1.02 1.06 219 368 219
(0.63, 2.05) (0.89, 2.00) (0.87, 1.42) (180, 254) (329, 402) (195, 298)
Placebo
(0.59, 1.49) (0.94, 1.60) (0.87, 1.35) (185, 249) (289, 451) (191, 296)
(0.62, 1.62) (0.96, 1.56) (0.90, 1.49) (186, 242) (300, 417) (225, 305)
(0.72, 1.62) (0.84, 1.74) (0.90, 1.30) (188, 243) (312, 420) (189, 282)
0.028 0.935 0.336 0.608 0.937 0.109
*CRP, C-reactive protein; IL-6, interleukin-6; TNF-a, tumor necrosis factor-a; sICAM-1, soluble intracellular adhesion molecule-1; sVCAM-1 soluble vascular cell adhesion molecule-1; MCP-1, monocyte chemotactic protein-1. y P values are shown for the treatment effect analysed using a mixed model analysis of covariance.
No significant changes in body weight (P Z 0.48), systolic BP (P Z 0.80), diastolic BP (P Z 0.64) and were observed following the intervention study. Furthermore, no significant differences were observed in energy (P Z 0.56), fat (P Z 0.86), SFA (P Z 0.75), monounsaturated fatty acid (P Z 0.70), polyunsaturated fatty acid (PUFA) (P Z 0.88), protein (P Z 0.40), carbohydrate (P Z 0.35), total fibre (P Z 0.96), dietary cholesterol (P Z 0.61), and alcohol (P Z 0.60) intake calculated at baseline and week 6 of the intervention and placebo periods. These results have been published previously in more details [7].
Discussion We have shown that 6-week daily consumption of a muffin enriched with a lignan complex isolated from flaxseed providing 500 mg SDG caused a significant difference in CRP concentration compared to a low-fat muffin with no lignans added among apparently healthy postmenopausal woman. The difference in plasma CRP concentration was caused by a higher CRP concentration following the placebo period compared to the lignan complex intervention period. No effect was observed on plasma concentrations of the other investigated pro-inflammatory proteins and adhesion molecules. The results from the present study are difficult to interpret due to a rise in CRP concentration during both the lignan complex and placebo period and several factors needs to be discussed. First, the results from the present study was analysed using a mixed model analysis of covariate with CRP baselines as covariate. Using this model, results were tested for a change in CRP following the lignan complex intervention period compared to the placebo intervention period at 6 weeks. The results showed that CRP concentrations were reduced following the lignan complex period compared to the placebo intervention period at 6 weeks and that the reduction was significant and approximately 15%. Therefore, the results suggest that daily consumption for 6 week of a low-fat muffin enriched with a lignan complex may reduce CRP concentrations compared to a low-fat muffin with no lignans added. Second, the change in CRP and IL-6 does not follow the same pattern in the present study, which would normally have been expected. CRP is primarily secreted in response
to IL-6, although an IL-6 independent regulation of CRP has been described [16,17]. In the present study, we did not observe any significant difference in IL-6 and TNF-a concentrations between the lignan complex intervention period compared to placebo. However, it has been shown that changes in CRP and IL-6 concentation does not necessarily follow the same pattern. Current use of hormone replacement therapy (HRT) has been associated with elevated serum CRP concentrations, whereas no similar association has been observed between current use of HRT and serum IL-6 concentrations [23]. Therefore, it is possible that the difference in plasma CRP concentration between the lignan complex intervention period and placebo period may be mediated through other systemic or local inflammatory pathways not examined in this study. Third, it is possible that the difference in CRP concentration following the lignan complex intervention period is a chance finding. However, all possible measures have been made in order to ensure valid results. Blood samples has been analysed using the same assay for each inflammatory marker in order to minimize variation. Furthermore, after the presented results on CRP were available, additional blood samples were analysed for CRP to make sure that the results was not caused by analytical errors. Results from the second analysis of CRP and the following statistical analysis provided similar results (data not shown). To our knowledge, no other study has examined the effect of plant lignans on inflammatory markers. A few studies suggest that isoflavones may reduce CRP [24] and have no effect on inflammation [25]. In addition, isoflavones may reduce the expression of sVCAM-1 [24,26] and have no effect on sICAM-1 and sVCAM-1 [27,28]. Results have show that isoflavones may affect markers of inflammation during a relatively short period of 6e8 weeks [24,26]. Plant lignans belong to the group of phyto-oestrogens as isoflavones. Therefore, it is reasonable to assume that markers of inflammation can be affected over a period of 6 weeks in the present study. However, it should be noted that both the above mentioned studies reporting an effect of isoflavones on markers of inflammation included a larger group of subjects [24,26] compared to the 23 women included in the present study. The relatively small sample size is a limitation of the present study. Furthermore, the women in our study were apparently healthy postmenopausal women with an initial TC concentration of
Flaxseed lignans and inflammatory markers 5.97 1.02 mmol/L. It may be interesting to examine whether a different treatment effect of the lignan complex would have occurred in hyperlipidemic women or women with a different CVD risk profile. In the present study, we measured 6 different markers of inflammation, but we only observed a significant effect on CRP. It is likely that the effect on CRP is caused by the increased serum ENL concentrations following the lignan complex intervention period. However, the mechanism of this effect is unknown. One suggested mechanism could be that SDG and its metabolites, END and ENL, may affect CRP concentrations due to their antioxidant activity, which have been shown in different in vitro model systems [12,13,29,30]. However, despite the antioxidant activity of SDG and its metabolites measured in vitro, previously results did not indicate a protective effect of SDG on susceptibility of serum lipoprotein to oxidation ex vivo [7]. It is therefore likely that other mechanisms may be responsible for the reduction in CRP. Expression of cellular adhesion molecules (CAMs) on the vascular endothelium is induced by pro-inflammatory cytokine such as IL-6 and TNF-a [22]. CRP has been shown to induce expression of ICAM-1, VCAM-1, and MCP-1 in vitro [20,21]. We found no changes in sICAM-1, sVCAM-1 and MCP-1 between the two intervention periods despite a significant difference in plasma CRP concentrations. These findings thus do not suggest that plant lignans affect expression of CAMs. Furthermore, the findings do not suggest a regulatory effect of CRP on the expression of CAMs. In conclusion, we have shown that 6-week daily consumption of a low-fat muffin enriched with a lignan complex isolated from flaxseed providing 500 mg SDG caused a significant difference in CRP concentration compared to a low-fat muffin with no lignans added among apparently healthy postmenopausal woman. The difference in CRP concentrations was independent of changes in other inflammatory markers. The lignan complex may have specific effects related to inflammation mediated through systemic or local inflammatory pathways not examined in this study. Further studies are needed in order to understand the importance of the reduction in CRP concentration.
Acknowledgement Thank to Ms. Helene Hausner for help during conduction of the study, and Dr. Ole Hels for help during the preparation of the manuscript, and Ms. Pia L Madsen, Ms. Yvonne Rasmussen, Ms. Berit Hoielt, Ms. Hanne Jensen, and Mr. Leif S Jakobsen for excellent technical support. The present study was carried out with financial supported from 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 Co (Decatur, IL, USA) funded the CRP and sICAM-1 analysis and provided the lignan complex.
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The effect of a lignan complex isolated from flaxseed on inflammation markers in healthy postmenopausal women ¨gel a, T. Tholstrup a, J.M. Bruun c J. Hallund a,*, I. Tetens b, S. Bu a
Department of Human Nutrition, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg C, Denmark b National Food Institute, Technical University of Denmark, Søborg, Denmark c Department of Endocrinology and Metabolism C, Aarhus University Hospital, Aarhus C, Denmark Received 13 November 2006; received in revised form 30 April 2007; accepted 2 May 2007
KEYWORDS Phyto-oestrogens; Lignans; Flaxseed; Enterolactone; Inflammation
Abstract Background and aim: Plant lignans are metabolised by the colonic micro-flora to the mammalian lignans enterodiol and enterolactone, which are hypothesized to be cardioprotective. The aim of this study was to investigate the effects of a plant lignan complex isolated from flaxseed, providing 500 mg/d of secoisolariciresinol diglucoside, on inflammatory markers. Methods and results: Healthy postmenopausal women (n Z 22) completed a randomised double-blind, placebo-controlled crossover study. Women consumed daily a low-fat muffin, with or without a lignan complex, for 6 weeks, separated by a 6-week washout period. A significant difference of approximately 15% (P Z 0.028) was observed for C-reactive protein (CRP) concentration between the lignan complex intervention period and placebo period. CRP concentrations (median; 25th, 75th percentiles) were 0.88 (0.63, 2.05) mg/L at baseline and 0.92 (0.59, 1.49) mg/L after the lignan complex intervention period compared with 0.80 (0.62, 1.62) mg/L at baseline and 1.10 (0.72, 1.62) mg/L after placebo. No significant differences in interleukin-6, tumor necrosis factor-a, soluble intracellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, and monocyte chemoattractant protein-1 were found between the lignan complex intervention period and placebo period. Conclusion: Daily consumption for 6 week of a low-fat muffin enriched with a lignan complex may reduce CRP concentrations compared to a low-fat muffin with no lignans added. ª 2007 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: þ45 3528 2485; fax: þ45 3528 2483. E-mail address: [email protected] (J. Hallund). 0939-4753/$ - see front matter ª 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.numecd.2007.05.007
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Introduction Flaxseed is the richest dietary source of the plant lignan secoisolariciresinol diglucoside (SDG), which is metabolised to the mammalian lignans enterodiol and enterolactone (ENL) by colonic bacteria [1]. Plant lignans belong to the group of phyto-oestrogens, which have chemical structures resembling those of endogenous oestrogen and which have binding affinity to the sex steroid-binding globulin [2]. Recently, a number of observational studies have suggested that mammalian lignans may have a protective effect against the risk of cardiovascular disease (CVD) [3e5]. High serum ENL concentrations have prospectively been associated with a reduced risk of acute cardiovascular events and lower coronary heart disease (CHD) and CVDrelated mortality in healthy men [3,6]. We have earlier shown that daily consumption for 6 week of a low-fat muffin enriched with a lignan complex significantly increases serum ENL concentrations and urinary ENL excretion in healthy postmenopausal women, but have no effect on plasma lipid concentrations, serum lipoprotein oxidation resistance, plasma antioxidant capacity [7] or endothelial function [8]. However, a number of controlled clinical intervention studies have shown that whole and defatted flaxseed lowers serum cholesterol in normal and hyperlipidemic subjects [9e11]. Furthermore, isolated SDG [12] as well as a lignan complex isolated from flaxseed [13] have been shown to reduce total cholesterol (TC), LDL cholesterol (LDL-C), and increases HDL cholesterol (HDL-C) in hypercholesterolemic rabbits, and reduce the development of hypercholesterolemic atherosclerosis by 34e73%. To our knowledge, no studies have so far examined the role of plant or mammalian lignans in the inflammation process. Chronic low-grade inflammation and increased adhesiveness for circulating leukocytes by vascular endothelial cells play a key role in the inflammatory process leading to atherosclerosis and CVD [14]. Increased plasma concentrations of the acute-phase reactant C-reactive protein (CRP) have been shown to be a strong predictor of CVD and CHD in apparently healthy women [15], but the underlying mechanism of this association is not understood. CRP is secreted by several cell-types, but primarily hepatocytes in response to IL-6 as well as other pro-inflammatory proteins [16e18]. Recent data suggest that CRP may also be produced in human coronary artery smooth muscle cells and the atherosclerotic lesion [18,19] indicating CRP as an active player in the development of cardiovascular disease. In vitro studies have shown that CRP induce increased expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) in human umbilical vein and coronary artery endothelial cells [20,21]. These studies suggest that CRP may be involved in the pathogenesis of atherosclerosis or inflammation. The adhesion of leukocytes to the vascular endothelium is mediated by cellular adhesion molecules (CAMs), such as ICAM-1 and VCAM-1, and is probably the first step in the atherosclerotic process. Following adhesion of leukocytes to the vascular endothelium, chemoattracting proteins (chemokines), such as MCP-1, play a key role in the migration of the leukocytes into the arterial intima [22].
J. Hallund et al. To our knowledge, no other studies have been published in relation to the possible role of the plant lignan SDG on central markers related to the inflammation process. The main objectives of this study are to determine whether a lignan complex isolated from flaxseed would affect plasma concentration of CRP, IL-6, TNF-a, sICAM-1, sVCAM-1 and MCP-1 in healthy post-menopausal women.
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 months). Prior to the study none of the women had used hormone replacement therapy in the past six months, or fatty acid, isoflavones, vitamin or mineral containing supplements in the past two months, or antibiotics in the past three months. None of the women had any history of diabetes, inflammatory diseases or CVD and they did not use any antihypertensive, anti-inflammatory or lipid lowering drugs. All women were non-smokers and had a BP of less than 160/90 mmHg. Screening blood samples were taken before entry and all subjects had TC <8 mmol/L, TAG <3 mmol/L, hemoglobin >7 mmol/L, and fasting glucose <6.5 mmol/L. A total of twenty-three women were included in the study. One woman withdrew from the study due to the use of antibiotics during the study period. Twenty-two women completed the study according to the protocol. Ethical approval was obtained from the Local Research Ethical Committee of Copenhagen and Frederiksberg (KF 01-130/02). All women received oral and written information about the study before they gave written informed consent.
Study design We performed a randomised, double blind, placebo controlled, crossover study. The women were instructed to consume their habitual diets throughout the study period but avoid any flaxseed consumption. Besides the habitual diet they consumed a low fat muffin daily, with or without a lignan complex, for 6 weeks, separated by a 6-week washout period. The women were instructed to consume the muffin 1 h after the evening meal 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: energy 642 kJ; protein 2.6 g; carbohydrate 34.1 g; fat 0.5 g and fibre 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. Compliance was assessed using study diaries, as well as ENL concentrations in serum and urine.
Flaxseed lignans and inflammatory markers Frozen muffins were handed out to the women every second week from the department for consumption at home. In addition, the women visited the department for five examinations during the study: one week before the beginning of the study (week 1); at the beginning of the study (week 0); after the first intervention period (week 6); at the beginning of the second intervention period after 6week washout period (week 12); and after the second intervention period (week 18). Height was measured at week 0. Body weight, blood pressure (BP), twenty-four hour urinary ENL excretion, and plasma concentrations of CRP, IL-6, TNF-a, sICAM-1, sVCAM-1 and MCP-1 were measured at week 0, 6, 12 and 18. Twelve hour fasting blood samples were collected during the morning after 15 min of supine rest. 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 A fasting blood sample was drawn into a 5 mL EDTA tube (Becton Dickinson #366457) for the analysis of high sensitive CRP (hsCRP). Thereafter, it was centrifuged at 1600 g for 10 min and stored at 20 C until further analysis. HsCRP was measured with a chemiluminescent immunometric assay (Diagnostic Products Corporation) using an Immulite 1000 analyser (Diagnostic Products Corporation). Intra- and inter-assay coefficient of variance was 3.4% and 3.7%, respectively. A fasting blood sample was drawn into a 10mL Na heparin vacutainer (Becton Dickinson #366480) for the IL-6, TNF-a, sICAM1, sVCAM-1 and MCP-1 analysis. Blood was centrifuged at 1600 g for 10 min at 4 C and plasma was stored at 80 C until analysis. IL-6, TNF-a, sICAM-1, sVCAM-1 and MCP-1 concentrations were determined using human IL-6 (Quantikine HS600, R&D Systems Europe Ltd., Abingdon, UK), TNF-a (Quantikine HSTA00C, R&D Systems Europe Ltd., Abingdon, UK), sICAM-1 (Parameter BBE1B, R&D Systems Europe Ltd., Abingdon, UK), sVCAM-1 (Parameter BBE3, R&D Systems Europe Ltd., Abingdon, UK) and MCP-1 (DuoSet DY297, R&D Systems Europe Ltd., Abingdon, UK) immunoassay kits. Intra- and inter-assay CVs were 5.5% and 7.9%, 3.5% and 6.9%, 6.4% and 5.0%, 8.3% and 9.5% and 8.1% and 9.6% for IL-6, TNF-a, sICAM-1, sVCAM-1 and MCP-1, respectively. Serum and urine ENL concentrations were determined using time-resolved fluoroimmunoassay (Labmaster Diagnostics, Turku, Finland) as previously described [7].
499 as means SEM. Data on the outcome of the study are expressed as the median and 25th, 75th percentiles. Data were analysed in SAS 8.02 (ª SAS Institute Inc., Cary, USA) 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 the baseline measurements for each period 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 were included to test if the treatment effect of the lignan complex varied according to the baseline values of the covariate. All data were logarithmictransformed to obtain normally distributed residuals. Differences were considered significant when P < 0.05.
Results The baseline characteristics of the women were as follows (mean SD): age 61 7 y, BMI 24.1 3.4 kg/m2, TC 5.97 1.02 mmol/L, LDL cholesterol 3.77 1.01 mmol/L, HDL cholesterol 1.75 0.45 mmol/L, TAG 0.98 0.26 mmol/L, systolic BP 124 13 mmHg, and diastolic BP 75 8 mmHg. Compliance assessed using study diaries showed that 98% of all muffins were consumed during the two intervention periods. A significant difference between the lignan complex intervention period and placebo period was observed for plasma CRP concentration (P Z 0.028), which was caused by a higher CRP concentration following the placebo period (Fig. 1). CRP concentrations were 0.88 (0.63, 2.05) mg/L at baseline and 0.92 (0.59, 1.49) mg/L after the lignan complex intervention period compared with 0.80 (0.62, 1.62) mg/L at baseline and 1.10 (0.72, 1.62) mg/L after the placebo period (P Z 0.028) (Table 1). There were no significant differences in plasma IL-6, TNF-a, VCAM-1, ICAM-1, and MCP-1 concentrations after the two intervention periods. ENL concentrations in serum and urinary ENL excretion increased from 46 8 nmol/L and 17 2 mmol/d, respectively, at baseline to 385 67 nmol/L (P < 0.001) and 94 11 mmol/d (P < 0.001) respectively, following the lignan complex intervention period. No changes in serum ENL concentrations or urinary ENL excretion were observed during the placebo intervention period. 6,00 5,00
Power calculations mg/L
4,00
The number of women needed in this study was calculated using the method of least standardized difference. 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 SD of the study outcome measures.
3,00 2,00 1,00 0,00
Statistical analysis Data describing the characteristics of the volunteers are summarized as the means SD. Data on ENL are expressed
Week 1
Week 6
Lignan complex
Week 1
Week 6
Placebo
Figure 1 Individual plasma CRP concentration during the lignan complex and placebo intervention period.
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J. Hallund et al.
Table 1 Plasma concentrations of circulation markers of inflammation in healthy postmenopausal women at baseline (week 0) and week 6 of the lignan complex intervention and placebo periods (n 22) (median values with their 25th and 75th percentiles) Py
Variable*
Lignan complex Baseline
Week 6
Baseline
Week 6
CRP (mg/L) IL-6 (ng/L) TNF-a (ng/L) SICAM-1 (ng/mL) SVCAM-1 (ng/mL) MCP-1 (ng/L)
0.88 1.28 1.08 204 349 238
0.92 1.14 1.07 212 352 226
0.80 1.16 1.06 205 363 255
1.10 1.02 1.06 219 368 219
(0.63, 2.05) (0.89, 2.00) (0.87, 1.42) (180, 254) (329, 402) (195, 298)
Placebo
(0.59, 1.49) (0.94, 1.60) (0.87, 1.35) (185, 249) (289, 451) (191, 296)
(0.62, 1.62) (0.96, 1.56) (0.90, 1.49) (186, 242) (300, 417) (225, 305)
(0.72, 1.62) (0.84, 1.74) (0.90, 1.30) (188, 243) (312, 420) (189, 282)
0.028 0.935 0.336 0.608 0.937 0.109
*CRP, C-reactive protein; IL-6, interleukin-6; TNF-a, tumor necrosis factor-a; sICAM-1, soluble intracellular adhesion molecule-1; sVCAM-1 soluble vascular cell adhesion molecule-1; MCP-1, monocyte chemotactic protein-1. y P values are shown for the treatment effect analysed using a mixed model analysis of covariance.
No significant changes in body weight (P Z 0.48), systolic BP (P Z 0.80), diastolic BP (P Z 0.64) and were observed following the intervention study. Furthermore, no significant differences were observed in energy (P Z 0.56), fat (P Z 0.86), SFA (P Z 0.75), monounsaturated fatty acid (P Z 0.70), polyunsaturated fatty acid (PUFA) (P Z 0.88), protein (P Z 0.40), carbohydrate (P Z 0.35), total fibre (P Z 0.96), dietary cholesterol (P Z 0.61), and alcohol (P Z 0.60) intake calculated at baseline and week 6 of the intervention and placebo periods. These results have been published previously in more details [7].
Discussion We have shown that 6-week daily consumption of a muffin enriched with a lignan complex isolated from flaxseed providing 500 mg SDG caused a significant difference in CRP concentration compared to a low-fat muffin with no lignans added among apparently healthy postmenopausal woman. The difference in plasma CRP concentration was caused by a higher CRP concentration following the placebo period compared to the lignan complex intervention period. No effect was observed on plasma concentrations of the other investigated pro-inflammatory proteins and adhesion molecules. The results from the present study are difficult to interpret due to a rise in CRP concentration during both the lignan complex and placebo period and several factors needs to be discussed. First, the results from the present study was analysed using a mixed model analysis of covariate with CRP baselines as covariate. Using this model, results were tested for a change in CRP following the lignan complex intervention period compared to the placebo intervention period at 6 weeks. The results showed that CRP concentrations were reduced following the lignan complex period compared to the placebo intervention period at 6 weeks and that the reduction was significant and approximately 15%. Therefore, the results suggest that daily consumption for 6 week of a low-fat muffin enriched with a lignan complex may reduce CRP concentrations compared to a low-fat muffin with no lignans added. Second, the change in CRP and IL-6 does not follow the same pattern in the present study, which would normally have been expected. CRP is primarily secreted in response
to IL-6, although an IL-6 independent regulation of CRP has been described [16,17]. In the present study, we did not observe any significant difference in IL-6 and TNF-a concentrations between the lignan complex intervention period compared to placebo. However, it has been shown that changes in CRP and IL-6 concentation does not necessarily follow the same pattern. Current use of hormone replacement therapy (HRT) has been associated with elevated serum CRP concentrations, whereas no similar association has been observed between current use of HRT and serum IL-6 concentrations [23]. Therefore, it is possible that the difference in plasma CRP concentration between the lignan complex intervention period and placebo period may be mediated through other systemic or local inflammatory pathways not examined in this study. Third, it is possible that the difference in CRP concentration following the lignan complex intervention period is a chance finding. However, all possible measures have been made in order to ensure valid results. Blood samples has been analysed using the same assay for each inflammatory marker in order to minimize variation. Furthermore, after the presented results on CRP were available, additional blood samples were analysed for CRP to make sure that the results was not caused by analytical errors. Results from the second analysis of CRP and the following statistical analysis provided similar results (data not shown). To our knowledge, no other study has examined the effect of plant lignans on inflammatory markers. A few studies suggest that isoflavones may reduce CRP [24] and have no effect on inflammation [25]. In addition, isoflavones may reduce the expression of sVCAM-1 [24,26] and have no effect on sICAM-1 and sVCAM-1 [27,28]. Results have show that isoflavones may affect markers of inflammation during a relatively short period of 6e8 weeks [24,26]. Plant lignans belong to the group of phyto-oestrogens as isoflavones. Therefore, it is reasonable to assume that markers of inflammation can be affected over a period of 6 weeks in the present study. However, it should be noted that both the above mentioned studies reporting an effect of isoflavones on markers of inflammation included a larger group of subjects [24,26] compared to the 23 women included in the present study. The relatively small sample size is a limitation of the present study. Furthermore, the women in our study were apparently healthy postmenopausal women with an initial TC concentration of
Flaxseed lignans and inflammatory markers 5.97 1.02 mmol/L. It may be interesting to examine whether a different treatment effect of the lignan complex would have occurred in hyperlipidemic women or women with a different CVD risk profile. In the present study, we measured 6 different markers of inflammation, but we only observed a significant effect on CRP. It is likely that the effect on CRP is caused by the increased serum ENL concentrations following the lignan complex intervention period. However, the mechanism of this effect is unknown. One suggested mechanism could be that SDG and its metabolites, END and ENL, may affect CRP concentrations due to their antioxidant activity, which have been shown in different in vitro model systems [12,13,29,30]. However, despite the antioxidant activity of SDG and its metabolites measured in vitro, previously results did not indicate a protective effect of SDG on susceptibility of serum lipoprotein to oxidation ex vivo [7]. It is therefore likely that other mechanisms may be responsible for the reduction in CRP. Expression of cellular adhesion molecules (CAMs) on the vascular endothelium is induced by pro-inflammatory cytokine such as IL-6 and TNF-a [22]. CRP has been shown to induce expression of ICAM-1, VCAM-1, and MCP-1 in vitro [20,21]. We found no changes in sICAM-1, sVCAM-1 and MCP-1 between the two intervention periods despite a significant difference in plasma CRP concentrations. These findings thus do not suggest that plant lignans affect expression of CAMs. Furthermore, the findings do not suggest a regulatory effect of CRP on the expression of CAMs. In conclusion, we have shown that 6-week daily consumption of a low-fat muffin enriched with a lignan complex isolated from flaxseed providing 500 mg SDG caused a significant difference in CRP concentration compared to a low-fat muffin with no lignans added among apparently healthy postmenopausal woman. The difference in CRP concentrations was independent of changes in other inflammatory markers. The lignan complex may have specific effects related to inflammation mediated through systemic or local inflammatory pathways not examined in this study. Further studies are needed in order to understand the importance of the reduction in CRP concentration.
Acknowledgement Thank to Ms. Helene Hausner for help during conduction of the study, and Dr. Ole Hels for help during the preparation of the manuscript, and Ms. Pia L Madsen, Ms. Yvonne Rasmussen, Ms. Berit Hoielt, Ms. Hanne Jensen, and Mr. Leif S Jakobsen for excellent technical support. The present study was carried out with financial supported from 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 Co (Decatur, IL, USA) funded the CRP and sICAM-1 analysis and provided the lignan complex.
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