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* Department of Social and Preventive Medicine,
Department of Exercise and Nutrition Sciences,
** Department of Medicine and
School of Nursing, University at Buffalo, Buffalo, NY and
Epidemiology Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy
2To whom correspondence should be addressed. E-mail: muti{at}buffalo.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • postmenopausal women • diet • serum phytosterols
Plant sterols or phytosterols are common components of plant foods, especially plant oils, seeds and nuts, cereals and legumes (1). The most common phytosterols are campesterol, ß-sitosterol and stigmasterol. Structurally, these compounds are similar to cholesterol, except for an additional hydrocarbon chain at the C-24 position. The human body does not synthesize phytosterols endogenously. Circulating phytosterols are derived exclusively through intestinal absorption (2). Serum phytosterol levels in humans range from 7 to 41 µmol/L (2.9 to 17.0 mg/L) (3).
Phytosterols may have anticarcinogenic properties. At concentrations of 16 µmol/L (6.7 mg/L), phytosterols inhibit growth and induce apoptosis in human prostate cancer cells (4). In in vivo experiments, rats fed a diet supplemented with 0.3% ß-sitosterol had a lower incidence of chemically induced tumors than controls (5). Awad et al. (6) observed that ß-sitosterol inhibits the growth of MBA-MD-231, a human breast cancer line. In addition, recent studies indicated that feeding SCID (severe combined immunodeficient) mice a diet supplemented with phytosterols for 8 wk inhibits the growth of human breast cancer by 33% and reduces metastasis by 20% (7). Similar results have also been obtained using PC3 cells, a human prostate cell line (8).
Populations at low breast cancer risk consume more dietary phytosterols than those at high risk. For example, the Japanese consume a plant-based food and low animal fat diet that is rich in phytosterols. Their diet concentrations range from 8 to 12 µmol/L (3.4 to 5.0 mg/L). The Japanese also have a low incidence of breast cancer (9,10). In contrast, high breast cancer incidence populations in Western countries, such as the United States and Northern Europe (including Northern Italy), have low vegetable and high fat intakes which are associated with low dietary phytosterol of 2–6 µmol/L (80–250 mg/d) (9–11). Thus, phytosterol intake may explain in part the protective effect of a vegetable-rich diet on breast cancer incidence that has been observed in some epidemiological studies (12–13).
The aim of the present study was to evaluate the effect of diet, and particularly the effect of a plant food–based diet, low in animal fats, on serum phytosterol levels in hyperandrogenic postmenopausal women who are characterized by a high risk of breast cancer because of their endocrine profile (14). We hypothesized that a plant-based food would increase serum levels of phytosterols, in particular campesterol and ß-sitosterol, in a convenience sample of women.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The data for this study came from a randomized controlled trial conducted in Milan, Italy from November 1995 to November 1996, the Diet and Androgens Study (DIANA)2 (15). The Division of Epidemiology at the Istituto Nazionale Tumori (Italian National Cancer Institute), Milan, Northern Italy conducted the DIANA study to evaluate the effect of a plant food–based diet, rich in vegetables and fibers and low in animal fats and refined carbohydrate, on endogenous hormones, particularly serum androgens, the sex hormones that bind globulin and insulin. In October 1995, we recruited participants through Italian newspaper and TV advertisements for this trial on diet and women’s health in Milan (one of the largest industrial cities in Italy). Eligibility criteria were: 1) absence of menstrual cycles for at least 2 y before enrolling in the study; 2) no current or previous (last 6 mo before recruitment in the study) hormonal treatment; 3) no history of bilateral ovariectomy; 4) no personal history of cancer; 5) no current adherence to a vegetarian or macrobiotic diet or to any other medically prescribed diet; and 6) no current treatment for diabetes mellitus. We recruited a total of 312 women. All women provided written informed consent and the Scientific and Ethical Committee of the Istituto Nazionale Tumori in Milan approved the study.
After fasting, participants’ blood samples were collected between 0700 and 0900 h. Serum testosterone was measured in all 312 participants. We considered a total of 104 women in the upper tertile of serum testosterone (testosterone 13.18 pmol/L) to be hyperandrogenic and selected these women for the present study because they are at an increased risk for breast cancer (14,16). The hyperandrogenic women were randomized into either an intervention or a control group (52 women each), within blocks of age (above or under 58 y, the median age), of prebaseline testosterone levels (categorized in three levels) and of prebaseline insulin (three levels).
Women randomized to the dietary intervention group were instructed to follow the diet described below for 18 wk, from February to June 1996. The control group received only a general recommendation to increase vegetable and fruit consumption. At baseline (January 1996) and at the end of the intervention period (June 1996), blood samples taken after fasting and 24-h urine samples were collected between 0700 and 0900 h from all 104 participants. Serum and urine samples were stored at -80°C.
Dietary intervention.
Women in the dietary intervention group participated twice weekly in workshops on how to cook with vegetables during the intervention period. Subjects prepared and consumed study meals during each workshop.
A typical meal included either a vegetable soup or some fresh vegetables, occasionally including seaweed, and a main dish with whole rice or pasta or other whole cereals accompanied by legumes, cruciferous vegetables, other cooked vegetables or fish. We advised women to consume at least one serving of a soy product everyday, e.g., beans, tofu, tempeh, soymilk or miso. Emphasis was placed on the preparation of sweets and cookies with raisin, fruit juice or naturally fermented rice or barley malt instead of sugar, with flaxseeds or other seeds instead of butter, and with soymilk instead of cow’s milk. Women were also instructed on how to substitute vegetable sources rich in protein and calcium for meat, eggs and dairy products (each to be consumed no more than once a week). In addition, participants were asked to avoid refined carbohydrates (sucrose, white bread and pastries based on refined flour) and to limit salt, but were encouraged to season food with unrefined olive oil and various seeds (instead of dairy fats), and to consume fish. Every week, participants received a 1-kg loaf of bread made with whole wheat and 8% flaxseed (half whole seeds, half milled), occasionally mixed with oats or rye. Subjects consumed this diet ad libitum and were not advised to reduce food intake (Table 1).
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Before randomization, participants completed a food frequency questionnaire developed for the European Prospective Investigation into Cancer and Nutrition (EPIC) study (17). To monitor compliance with the dietary recommendations, participants recorded the frequency of consumption of selected foods in 24-h diaries. Women in the dietary intervention group completed 1-d diaries 24 times and women in the control group, 10 times. We estimated the mean absolute intakes of nutrients and energy in the two groups based on five repeated computer-assisted 24-h dietary recalls conducted during wk 11–16 of the study using software developed for the EPIC study (17). The system makes use of the Italian food composition database (18). Participants’ height and weight were measured at the beginning and end of the study.
Study compliance.
Fifty of the 52 women in the dietary intervention group completed the program. Two women completed only about half of the program. Overall, only five women were absent more than five times from the 36 workshops and did not eat the prepared meals. We excluded two women from the control group because they did not have a follow-up examination. In addition, three women in the control group did not have enough serum for the phytosterol determinations. Therefore, this study included data from 99 women: 52 in the dietary intervention and 47 in the control group.
Laboratory analysis.
For each replicate or sample, 0.5 mL serum was used. 5-
-Cholestane (10 µg; Sigma Chemical, St Louis, MO) was added in all replicates and samples as an internal standard, followed by saponification with 1 mol/L ethanolic KOH and heating at 80°C for 1 h. Sterols were extracted with hexane. GC was used to examine sterols without derivatization. The GC was equipped with a 0.9-m capillary column (model EC-5; Alltech, Deerfield, IL) and the temperatures were maintained at 265°C and 300°C for the oven and the injection port, respectively. Nitrogen was used as the carrier gas. Identification of the peaks was accomplished using the retention times of authentic sterol standards (Sigma Chemical). The areas under the peaks were integrated and corrected for recovery and response of the GC to different sterols using the internal standard. Serum cholesterol was measured using a certified cholesterol calibrator (Sigma Chemical). All determinations were conducted in duplicate.
Samples from the same subject (baseline and after the completion of the intervention) from both the intervention and the control groups were analyzed together by a laboratory technician who was unaware of treatment. Thus, because the within-run precision is an important source of technical variability, it was evaluated by analyzing unlabeled duplicates (from single clinical specimens collected from control subjects) placed at the beginning, in the middle and at the end of every laboratory run. The technical variability was expressed as the intraclass correlation coefficient (ICC), a correlation index that took into account both the intra- and interassay variability of the analytical tests. The ICC for ß-sitosterol was 0.88 (0.63, lower bound) and for campesterol 0.57 (0.15, lower bound).
Previously, we evaluated the intraindividual variability of ß-sitosterol and campesterol in a study conducted at the Department of Social and Preventive Medicine in 1998 (20). Seven premenopausal healthy women were recruited from the personnel of the department. Over a 6-mo period, each woman once a month after fasting provided a blood sample at the same time of day, and the same numerical day of her menstrual cycle. Serum was processed immediately after blood draw and stored at -80°C. All serum samples from the same individual were processed at the same time by the same technician at the end of the 16-mo period. The overtime intraindividual variability for campesterol and ß-sitosterol, expressed as ICC, were 0.58 (0.31 lower bound) and 0.91 (0.49 lower bound), respectively. These findings indicated a good to fair reliability of these determinations.
Statistical analysis.
The statistical analysis focused on changes in serum ß-sitosterol and campesterol concentrations, computed as the difference between baseline and end-of-study levels. Mean changes in the dietary intervention group were compared with those in controls by nonparametric analysis, the Wilcoxon rank sum test ( < 0.005). Serum phytosterol concentrations and body weights were statistically compared using the Student’s t test. SAS version 8.2 was used for the statistical analysis.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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As reported in other diet-recall studies (21), the interviews conducted from wk 11–16 of this study slightly underestimated the total energy intake with respect to energy requirement (21). However, the data showed a lower total energy intake in the intervention group than in the control group, a mean of 250 kcal/d (1045 kJ/d) (P < 0.004), mainly due to the lower intake of total and saturated fat. These results indicate that energy intakes of the subjects were lower than published energy requirements for the Italian population of postmenopausal women.
According to the food frequency diaries compiled during the study, the intervention group consumed meat or meat products twice a week compared with once a day in the control group, but consumed fish more often (3 times/wk versus 1.5 in controls). Milk and cheese consumption was cut by half (0.4 versus 1.0 servings/d) and butter was virtually eliminated. A soy product was consumed by the test group a mean of 1.7 times/d; flax seeds, either in bread or cookies or as such were eaten every day (about 8 g/d), and seaweed was consumed every other day as ingredients of various dishes. The control group rarely, if ever, consumed any of these food items. The intervention group also consumed the following much more often than controls: whole rice or other whole grain or whole-meal cereal products (2.5 versus 0.5 times/d), walnuts, almonds, sesame and other seeds (1.2 versus 0.05 times/d), legumes (0.5 versus 0.1 times/d), cruciferous vegetables (0.8 versus 0.1 times/d), and berries (0.4 versus 0.1 times/d). Other vegetables and fruits were consumed almost as frequently by the control group as by the intervention group (2.2 and 2.3 times/d, respectively).
The absolute change in serum total phytosterol concentrations were greater in the dietary intervention group than in the control group (Table 2). The percent change tended to differ (P = 0.06). However, only for ß-sitosterol did the absolute and percent changes within a group differ significantly between groups (P = 0.0017). We repeated the analyses adjusting for baseline concentrations of serum campesterol, ß-sitosterol and total phytosterols and the results did not differ from the unadjusted estimates (data not shown). We also adjusted the analyses for BMI, waist-to-hip ratio and cholesterol at baseline and the results did not differ from the unadjusted results (data not shown). Total cholesterol levels decreased more in the intervention group (-14%) than in the control group (-4%; P = 0.0005). The intervention group lost more weight (P < 0.0001) than the control group: 4.06 kg (range, -0.6–-8.8 kg) versus 0.54 kg (range, +2.2–5.3 kg).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Although there is evidence that phytosterols may have anticarcinogenic properties (22), the mechanism by which they inhibit tumor growth is not known. However, investigators have hypothesized that phytosterols may influence cancer development by altering cell signaling pathways, in particular the sphingomyelin (SM) cycle (4,23). The activation of this pathway by several agonists inhibits growth (25) and influences several other processes such as differentiation (25), cell-cell interaction (25) and apoptosis of tumor cells (23,26). The incorporation of phytosterols in a human cancer cell line reduces membrane SM, suggesting an activation of the SM cycle (27).
Despite the experimental and ecological evidence for a role of phytosterols in protection against breast cancer, there is insufficient information describing diet influence on serum phytosterol levels and how, in turn, phytosterols might influence breast cancer risk. There are two published studies of serum phytosterols in relation to breast cancer in humans. In the first, conducted on 10 women with benign breast disease and 11 breast cancer patients, phytosterol concentrations in adipose tissue (campesterol, stigmasterol and ß-sitosterol) were lower in breast cancer cases than in the controls, although the differences were not significant (28). In the second study on 11 healthy women, 7 lactating women and 14 women diagnosed with breast cancer, serum phytosterol levels did not differ among the groups (29). However, these studies had major limitations due to low internal validity (e.g., lack of information on comparability of cases and controls, lack of control of technical and biological variability of phytosterols for cases and controls) and to the small sample size. Furthermore, none of the studies included dietary information.
We observed that the levels of serum phytosterols in participants in this study ranged from 22 to 27 µmol/L (9.1 to 11.2 mg/L) which is similar to the 7–41 µmol/L range (2.9–17.0 mg/L) found in previous studies conducted on normal individuals (3,31). In the intervention group, ß-sitosterol increased 20% due to the intake of nuts, seeds and soy in the diet (1,31). In this study we observed higher variability in serum campesterol compared with ß-sitosterol. This may reflect: 1) the variation in the concentrations of these phytosterols in different vegetables and plant foods consumed (1,30); 2) the differences in their absorption and elimination rates from the body (9,30); 3) the slightly larger technical variability in our study for campesterol in comparison to ß-sitosterol; and 4) the larger intraindividual variability for campesterol in the previously conducted reliability study (20).
In conclusion, the present study supports the hypothesis that a plant-based diet rich in cereal fibers, soy and flaxseed can increase circulating levels of ß-sitosterol. These findings indicate that phytosterols can be used as biomarkers of exposure in observational studies or as compliance indicators in dietary intervention studies of cancer prevention.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: DIANA, diet and androgens study; EPIC, European Prospective Investigation into Cancer and Nutrition Study; ICC, intraclass correlation coefficient; SM, sphingomyelin.
Manuscript received 30 April 2003. Initial review completed 16 May 2003. Revision accepted 23 September 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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