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Endocrine Care |
Department of Nutritional Sciences (E.A.L., L.J.H., D.A.K., S.J., B.P.D., B.J.S., P.H.A.), Oklahoma State University, Stillwater, Oklahoma 74078; and Department of Obstetrics and Gynecology (R.D.W.), University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190
Address all correspondence and requests for reprints to: Bahram H. Arjmandi, Department of Nutritional Sciences, 416 Human Environmental Sciences, Oklahoma State University, Stillwater, Oklahoma 74078-6141. E-mail: . arjmand{at}okstate.edu
Abstract
The risk of cardiovascular disease and osteoporosis drastically increases at the onset of menopause. Phytoestrogens have been suggested to inhibit bone loss and protect the cardiovascular system, in part by improving lipid profiles. The purpose of the present study was to examine the effects of flaxseed, a rich source of the phytoestrogens called lignans, on lipid metabolism and biomarkers of bone turnover in postmenopausal women. Postmenopausal women who were not on hormone replacement therapy were assigned to one of two treatment groups in a double-blind randomized study. Women were asked to consume 40 g of either ground flaxseed or wheat-based comparative control regimen daily for 3 months. In addition, all subjects received 1,000 mg calcium and 400 IU vitamin D daily. Flaxseed supplementation lowered (P < 0.05) both serum total cholesterol and non-high-density lipoprotein cholesterol by 6%, whereas the comparative control regimen had no such effect. Flaxseed regimen reduced serum levels of both low-density- and high-density-lipoprotein cholesterol by 4.7% and triglyceride by 12.8%, albeit not statistically significant. Serum apolipoprotein A-1 and apolipoprotein B concentrations were significantly (P < 0.005) reduced by 6 and 7.5%, respectively, by the flaxseed regimen. Markers of bone formation and resorption were not affected by either of the treatments. The findings of this study indicate that flaxseed supplementation improves lipid profiles but has no effect on biomarkers of bone metabolism in postmenopausal women.
ALTHOUGH HORMONE REPLACEMENT therapy is efficacious in relieving postmenopausal symptoms such as hot flashes and vaginal dryness and in the prevention of bone loss, its cardiovascular protective effects are being questioned (1). High blood cholesterol level is a major risk factor for cardiovascular disease (2, 3). In addition to existing drug therapies, certain nutritional factors reduce serum cholesterol, including dietary fiber (4, 5, 6, 7, 8, 9), plant sterols (10, 11, 12), and phytoestrogens (13, 14, 15, 16, 17, 18). Among food sources rich in phytoestrogens, flaxseed has been reported to lower cholesterol in a limited number of human (19, 20, 21, 22, 23) and animal (24) studies.
Flaxseed is the richest food source of lignans, one of the major groups of phytoestrogens (25), and is increasingly being incorporated into human diets because of its reported health benefits. Lignans have been implicated as having anti-tumorigenic (26), estrogenic and/or anti-estrogenic (27), and antioxidant (28, 29, 30) properties. Prasad (24) reported that rabbits receiving secoisolariciresinol diglucoside, the major lignan found in flaxseed, had reduced hypercholesterolemic atherosclerosis that could be partly attributed to lower total- and low-density lipoprotein (LDL)-cholesterol concentrations. A recent population study also found an inverse association between serum lignan concentrations and the risk of acute coronary heart disease (31). However, the hypocholesterolemic effects of whole flaxseed can also be attributed to its 
-linolenic acid and fiber components (20, 21, 23). Therefore, the extent to which the individual components of flaxseed contribute to its cholesterol-lowering properties needs to be explored. Due to structural similarities between lignans and estrogen, it can be postulated that lignans present in flaxseed may also play a role in the maintenance of skeletal health. Hence, in this 3-month clinical study, in addition to the assessment of lipid parameters, the effects of flaxseed supplementation on selected blood and urinary markers of bone metabolism in postmenopausal women were investigated.
Subjects and Methods
Subjects
Postmenopausal women younger than 65 yr old who were not on hormone replacement therapy or any prescription medications known to influence lipid or bone metabolism were recruited. Women with cancer, liver disease, hypothyroidism or hyperthyroidism, gastrointestinal disorders, insulin-dependent diabetes mellitus, pelvic inflammatory disease, and endometrial polyps were excluded from the study. The study protocol was approved by the Institutional Review Boards at Oklahoma State University and the University of Oklahoma Health Sciences Center. Subjects signed a consent form after being provided with oral and written descriptions of the study. A complete medical history was obtained from all subjects before initiating the treatments. Subjects were also given routine physical and gynecological examinations including a vaginal smear, performed by an obstetrician and evaluated by a pathologist. Subjects lived at home, consumed their habitual diet, and maintained their usual physical activity.
Study design
Fifty-eight postmenopausal women were randomly assigned to one of two dietary treatments (n = 29 per treatment) in a controlled double-blind parallel study. The dietary treatments consisted of 40 g of either ground whole flaxseed or wheat-based comparative control regimen to be consumed daily for a period of 3 months. The macronutrient composition and the calcium and phosphorus contents of both regimens are shown in Table 1
. To provide some protection against rapid bone loss, all study participants were provided with 1,000 mg elemental calcium plus 400 IU vitamin D for daily consumption. The dietary regimens and calcium plus vitamin D supplements were distributed to the subjects on a monthly basis. Subjects were asked to return any unused supplements to monitor treatment compliance. The study participants were advised by a registered dietitian to make appropriate adjustments in their daily food consumption to account for the additional energy, fat, and protein intakes.
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Physical and gynecological examinations were repeated at the end of the study. Maturation index (MI) was calculated as the percentage of superficial cells plus half of the percentage of intermediate cells (32).
Overnight fasting blood was obtained at baseline and at the end of the study. Blood was centrifuged for 15 min at 1,500 x g, and serum aliquots were stored at -80 C until analyzed. Study participants were also instructed to collect 24-h urine before initiation of treatment and at the end of the study. Urine volume was recorded, and urine aliquots were stored at -20 C until analyzed.
Analytical methods
Serum total cholesterol and triglyceride concentrations were determined enzymatically using kits from Roche Diagnostics (Sommerville, NJ). Serum high-density lipoprotein (HDL) cholesterol was determined by a direct method (Unimate HDL Direct, Roche Diagnostics) that uses the combined action of polymers, polyanions, and detergent to solubilize cholesterol from HDL but not from very LDL, LDL, and chylomicrons. LDL-cholesterol concentration was calculated by the Friedewald equation (33). Non-HDL-cholesterol concentration was calculated by subtracting HDL cholesterol from total cholesterol. Apolipoprotein A-1 (apo A-1) and apolipoprotein B (apo B) were determined by immunoturbidimetry using kits from Roche Diagnostics. These tests were performed using a Cobas-Fara II clinical analyzer (Montclair, NJ). The intra- and interassay coefficients of variation (CVs) were 1.5 and 2.1%, 2.0 and 2.6%, 1.2 and 2.9%, 1.4 and 3.4%, and 1.4 and 5.2%, for total cholesterol, triglycerides, HDL cholesterol, apo A-1, and apo B, respectively.
RIA kits were used to analyze serum IGF-I (Nichols Institute Diagnostics, San Juan Capistrano, CA), IGF-binding protein (IGFBP)-3, 17ß-estradiol (E2), estrone (E1), FSH, and SHBG (Diagnostics Systems Laboratories, Inc., Webster, TX). Serum total alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activities and serum calcium were determined colorimetrically using kits from Roche Diagnostics. These tests were performed on a Cobas-Fara II clinical analyzer. Bone-specific AP (BSAP) activity in serum was quantified by immunoassay in a microtiter format (Metra Biosystems, Mountain View, CA). The intra- and interassay CVs were 3.0 and 8.4%, 3.0 and 1.0%, 6.5 and 9.7%, 5.6 and 11.1%, 2.7 and 6.8%, 3.4 and 8.7%, 1.9 and 2.8%, 2.7 and 8.3%, 1.2 and 2.3%, and 3.9 and 7.6% for IGF-I, IGFBP-3, E2, E1, FSH, SHBG, AP, TRAP, calcium, and BSAP, respectively.
Urinary creatinine was measured colorimetrically with a commercially available kit from Roche Diagnostics using a Cobas Fara II clinical analyzer. Urinary deoxypyridinoline (Dpd) was measured by competitive enzyme immunoassay in a microassay stripwell format (Metra Biosystems, Mountain View, CA). Urinary excretion of helical peptide, a peptide derived from the helical region of 1 chain of type I collagen, was assayed using a competitive enzyme immunoassay in a microassay stripwell format (Quidel Corporation, Mountain View, CA). The intra- and interassay CVs were 1.7 and 6.3%, 4.3 and 4.6%, and 6.5 and 8.6% for creatinine, Dpd, and helical peptide, respectively.
Statistical analyses
The data were analyzed using SAS (Version 6.11, SAS Institute, Inc., Cary, NC). ANOVA and least square means were calculated using PROC MIXED. Data are reported as least square mean ± SE, unless otherwise indicated; P < 0.05 was regarded as significant.
Results
Of the 58 postmenopausal women initially included in the study, only 36 women (20 receiving the flaxseed regimen and 16 receiving the wheat-based regimen) completed the study. Reasons for attrition included medical conditions that prevented continued inclusion into the study (one subject from the wheat regimen), time constraints (two subjects from the wheat regimen), gastrointestinal problems (three subjects from the flaxseed and six subjects from the wheat regimen), lack of palatability of the dietary regimen (six subjects from the flaxseed and three subjects from the wheat regimen), and unrelated personal reasons (one subject from the wheat regimen).
There were no significant differences in the baseline and final values of body weight and body mass index (BMI) among the subjects in either treatment group (Table 2). However, body weight (P = 0.092) and BMI (P = 0.072) tended to be higher after a 3-month supplementation with the wheat-based regimen. This was not observed among women in the flaxseed group.
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). Although flaxseed regimen reduced serum levels of LDL cholesterol by 4.7% and triglyceride by 12.8%, these decreases did not reach statistical significance. HDL-cholesterol concentrations were also somewhat (P = 0.091) lowered by flaxseed consumption. Apo A-1 and apo B concentrations were both significantly reduced as a result of flaxseed supplementation but were not affected by the wheat-based regimen. The changes from baseline values of the various serum lipid parameters were compared between flaxseed and the wheat-based regimens (Fig. 1). The response to treatment was significantly different between flaxseed and wheat supplementation for total cholesterol (P < 0.01), non-HDL cholesterol (P < 0.05), apo A-1 (P < 0.05), and apo B (P < 0.001).
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). Neither the flaxseed nor the wheat-based regimen produced estrogenic effects as assessed by serum levels of E1, E2, FSH, and SHBG (Table 6). Women on the flaxseed regimen had slightly (P = 0.09) lower MI after 3 months, whereas wheat-based supplement had no such effect.
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In this study, we have shown that supplementation of flaxseed to the diets of postmenopausal women can lower concentrations of serum total cholesterol and non-HDL cholesterol. These findings are in agreement with the findings of our earlier study (19) in which flaxseed given in the form of bread and muffins reduced total cholesterol in postmenopausal women. However, in the present study, LDL-cholesterol concentration was lowered by about 4.7% only vs. 14.7% in the previous study. Feeding the subjects whole ground flaxseed instead of its incorporation into baked products may have contributed to this difference. It has been suggested that the consumption of up to 50 g flaxseed in its raw form is safe (22), but it is not clear whether the constituents of flaxseed that influence lipid metabolism such as secoisolariciresinol diglucoside and -linolenic acid are as bioavailable when flaxseed is consumed in its raw form. However, whether processing or temperature will affect the bioavailability of flaxseed components requires further investigation.
In a crossover study in which hyperlipidemic men and postmenopausal women were fed 50 g of partially defatted flaxseed daily for 3 weeks, Jenkins et al. (23) observed overall reductions of 5.5 and 9.7% in serum total- and LDL-cholesterol concentrations, respectively. The investigators concluded that flaxseed gum is likely the major active ingredient responsible for the lipid-lowering action of flaxseed. Moreover, other constituents present in flaxseed may also play an essential role in lipid metabolism. For instance, the hypocholesterolemic effects of -linolenic acid have been reported in both animals (34) and humans (35). Garg et al. (34) demonstrated that feeding an -linolenic acid-rich diet to rats lowered serum cholesterol levels more effectively than a diet rich in linoleic acid. Clinical trials (35) have provided further evidence that consumption of -linolenic acid-rich oils such as flaxseed oil may offer greater protection against cardiovascular disease than linoleic acid-rich oils through their effects on platelet functions.
Similar to the findings of Jenkins et al. (23), we observed that flaxseed reduced serum levels of both apo B and apo A-1. However, the magnitude of change in serum apo B (10%) was greater than that of apo A-1 (6%), suggestive of cardioprotective properties of flaxseed. Apo B is a more sensitive indicator of the risk of heart disease than total cholesterol because it reflects the number of lipoproteins that are associated with the development of atherosclerosis such as LDL, very LDL, and chylomicron remnants (36).
Whether the hypolipidemic effects of whole flaxseed are due to a single component or the interactions among its components remains unclear. Kuroda et al. (37) evaluated the hypolipidemic properties of a series of diesters of arylnaphthalene lignans. They reported that these synthetic lignans effectively lower serum total cholesterol and LDL cholesterol while increasing HDL cholesterol. Lignans have also been shown to modulate activities of 7 -hydroxylase and acyl CoA cholesterol transferase (38), two of the key enzymes involved in cholesterol metabolism. Prasad et al. (39) concluded that reduction in hypercholesterolemic atherosclerosis by flaxseed is due to a decrease in serum total cholesterol and LDL cholesterol and that the antiatherogenic activity of flaxseed is independent of its -linolenic acid content. Soluble fiber mucilage present in flaxseed may also contribute to the observed hypocholesterolemic properties (4, 5). Hence, the mode of action of flaxseed is unclear and needs to be investigated in future studies.
As far as the effect of flaxseed on bone is concerned, there is a paucity of data. The findings of a study by Babu et al. (40) indicated that feeding whole or defatted flaxseed to weanling female rats for 56 d suppressed serum total AP activity, a nonspecific marker of bone formation. In that study (40), the indices of bone resorption were not assessed, leading us to speculate whether flaxseed or its lignans behave similarly to estrogen by suppressing both bone formation and bone resorption. It is conceivable that lignanic compounds, analogous to estrogen, also directly exert effects on estrogen-responsive tissues, including bone, through ERs. This notion is supported by more recent findings that human osteoclasts (41) and osteoblasts (42) express ERs, including ERß, the receptor to which phytoestrogens preferentially bind (15, 43). However, contrary to expectations, in the present study we have not seen any effects of flaxseed supplementation on indices of bone turnover.
In summary, the findings of the present study suggest that flaxseed consumption by postmenopausal women is effective in reducing total cholesterol, non-HDL cholesterol, and apo B, known risk factors of coronary heart disease. Additionally, flaxseed did not exert any estrogenic properties as assessed by unaltered circulating levels of sex hormones, SHBG, and MI. With respect to bone, the findings of this 3-month study indicate that flaxseed has no effect on bone metabolism, as evident by its lack of effects on biomarkers of bone turnover.
Acknowledgments
We thank Mr. Paul Stitt at the Natural Ovens of Manitowoc, Wisconsin, for providing the treatment regimens for this study.
Footnotes
This work was supported, in part, by NIH Grant R03-AG16487-01.
Abbreviations: AP, Alkaline phosphatase; apo A-1, apolipoprotein A-1; apo B, apolipoprotein B; BMI, body mass index; BSAP, bone-specific AP; CV, coefficient of variation; Dpd, deoxypyridinoline; E1, estrone; IGFBP, IGF binding protein; MI, maturation index; TRAP, tartrate-resistant acid phosphatase.
Received September 25, 2001.
Accepted December 21, 2001.
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