Rye bread intake improves oxidation resistance of LDL in healthy humans.

BACKGROUND: Oxidatively modified LDL particles contribute to atherogenic development and therefore dietary interventions for promoting oxidation resistance of LDL are of interest. The capacity of LDL to resist oxidation can be determined ex vivo by exposing isolated LDL particles to copper ions and measuring the formation of conjugated dienes by spectrophotometry. OBJECTIVE: The aim of this trial was to determine the effect of none versus high intake of rye bread on the oxidation resistance of LDL in healthy humans while otherwise on habitual diet. DESIGN: Sixty-three healthy subjects excluded rye products for one week (baseline), followed by a stepwise addition of rye bread from 99 g/d during the first two weeks to 198 g/d during the following two weeks. Additionally plant sterols were incorporated into the rye bread for half of the subjects to study cholesterol-lowering. The resistance of LDL against copper-induced oxidation was determined at baseline and at the end of the rye-period by monitoring formation of conjugated dienes. RESULTS: We observed a significant increase in the oxidation resistance of LDL, determined as a prolongation of the lag time (P < 0.001) and decrease in the slope of the propagation phase (P = 0.048) from baseline to the end of the rye-period without changes in vitamin E concentration. We observed no significant differences in the oxidation resistance of LDL between subjects who did or did not receive plant sterols. CONCLUSIONS: Rye bread intake improved significantly the oxidation resistance of LDL. Further studies are needed to clarify the protective mechanism(s).

Excretion of estrogens, catecholestrogens and phytoestrogens in carriers of BRCA1 gene mutations: effects of metformin.

BRCA1 gene mutation is associated with a combination of excessive aromatase activity/expression, predominantly estrogen receptor-negative phenotypes of tumors, and only scarce information about estrogen contents in body fluids. In the present work, isotope dilution capillary gas chromatography/mass spectrometry was used to study urinary excretion of estrogens, their catechol metabolites, and phytoestrogens in 22 women (11 with BCRA1 gene mutations and 11 without these mutations) in average 5.1+/-0.4 years before surgery for breast cancer. BCRA1 mutation carriers (including 3 premenopausal females) compared with respective controls showed significantly higher urinary estradiol and estrone excretion and a trend to an increased 2-OH-E2 excretion. In the subgroup of untreated postmenopausal women, BCRA1 mutation carriers showed a trend to increased estradiol and estrone excretion and to a higher value of the mean levels of all estrogen metabolites tested. The treatment after the baseline laboratory investigation of 6 women from postmenopausal group with the antidiabetic biguanide metformin for 3 months was associated with decreases in the excretion rates of 4-hydroxyestradiol, 2-methoxyestradiol, and 16-epiestriol and did not influence phytoestrogen excretion. The decrease in 2-methoxyestrogen excretion was more consistent in women without BCRA1 mutations than in BCRA1 mutation carriers. The data suggest the possibility that aromatase complex activation in BCRA1 mutation carriers is combined with increases in both, estrogen metabolism into catecholestrogens and their inactivation by methoxylation, and that metformin may affect both of these pathways.

Variation in fasting and non-fasting serum enterolactone concentrations in women of the Malmö Diet and Cancer cohort.

OBJECTIVES: The aim of this study was to examine the variation of enterolactone from fasting and non-fasting blood of middle-aged healthy women eating a normal diet to determine the usefulness of a single sample in epidemiological studies. SUBJECTS AND METHODS: Twenty-six women born between 1940 and 1950 were recruited within the Malmö Diet and Cancer cohort. Three non-fasting and two overnight fasting samples were collected from each individual during a 5-week period. Twenty-one participated in all measurements. Enterolactone concentrations were analyzed by time-resolved fluoroimmunoassay. RESULTS: The within-subject and between-subject variations (coefficient of variations, CV) were estimated to 59 and 89% respectively for fasting samples and 71 and 67% for non-fasting samples. The intraclass correlation coefficients (ICC) were estimated to 0.66 (95% confidence interval (CI) 0.35-0.84) for fasting and 0.48 (95% CI, 0.22-0.72) for non-fasting samples. CONCLUSIONS: Although the estimated ICC for blood samples was moderate, it indicates that enterolactone levels of both fasting and non-fasting blood samples should be useful in future projects within the Malmö Diet and Cancer cohort.

Urinary phytoestrogen excretion of rats bearing methylnitrosourea-induced mammary carcinoma in response to treatment with 2-methoxyestradiol.

The effect of treating mammary tumor-bearing rats with 2-methoxyestradiol (2-MeE2) on the urinary excretion of 12 phytoestrogens was investigated and compared with the changes in urinary excretion of estradiol metabolites. Alterations of excretion were registered for isoflavonoids, lignans and coumestans. However, due to large variations statistical significant differences were found only for two lignans, i.e. significant increases of enterodiol and matairesinol. Since the single components of phytoestrogens showed diverse alterations, excretions were expressed also by the ratio of total isoflavonoids to total lignans and compared with the estrogen ratios 2-hydroxyestrone to 16alpha-hydroxyestrone and A-ring to D-ring metabolites. The ratio of isoflavonoids to lignans was consistently decreased, whereas both ratios of estradiol metabolites were highly increased. The latter effect is probably due to demethylation of 2-methoxyestrone resulting in high catechol estrogen levels in urine. These results suggest that the high levels of catechol estrogens, produced by 2-MeE2 treatment, may have influenced the urinary excretion pattern of phytoestrogens.

Preliminary antiproliferative effects of some species of Terminalia, Combretum and Pteleopsis collected in Tanzania on some human cancer cell lines.

Methanolic extracts (25 microug/ml) of species belonging to the genera of Combretum, Terminalia and Pteleopsis, collected during a field expedition in Tanzania in 1999, were screened for their antiproliferative and cytotoxic effects against three human cancer cell lines (HeLa, cervical carcinoma; T 24, bladder carcinoma; and MCF 7, breast carcinoma). A leaf extract of Combretum fragrans and a fruit extract of C. zeyheri gave the strongest antiproliferative and cytotoxic effects of all the twenty-four extracts screened in this investigation. In contrast to the highly powerful leaf extract of C. fragrans, the root extract of this species gave no cytotoxic effects against the investigated cancer cell lines at a concentration of 25 microg/ml. The other investigated species of Combretum and Terminalia differed greatly in their cytotoxic potential. Root extracts of Terminalia sambesiaca and T. sericea gave the strongest cytotoxic effects of the five species of Terminalia used in this study. Eight of the twenty-four investigated plant extracts showed pronounced cytotoxic effects (<30% proliferation compared to the control) against the T 24 bladder cancer cells, seven against the HeLa cells and four against the MCF 7 cells.

No effect on adenoma formation in Min mice after moderate amount of flaxseed.

BACKGROUND & AIM: The mammalian lignan enterolactone (ENL) produced from plant lignans, e. g. secoisolariciresinol diglycoside (SDG), may protect against various cancers in humans. The present work aims to evaluate the effect of flaxseed on tumour formation in multiple intestinal neoplasia (Min) mice, a model for colon tumorigenesis. DESIGN: Male and female Min mice were fed either with a non-fibre control diet or the same diet supplemented with 0.5 % (w/w) defatted flaxseed meal. Conversion of SDG to the mammalian lignans enterodiol (END) and ENL in the gut, and plasma ENL, were measured by HPLC with coulometric electrode array detector (CEAD) and timeresolved fluoroimmunoassay, respectively. Wild-type mice were also fed with the experimental diets in order to see whether lignan metabolism is different in Min and wild-type mice. RESULTS: The total number of adenomas or their size in the small intestine was not different in the flaxseed and control groups. The flaxseed group had a tendency for a decreased number of colon adenomas in both genders. Gender and genotype based differences were found in the intestinal ENL levels. When compared to Min females, Min males in the flaxseed group had several fold higher ENL levels in the small intestine (Min males 125 +/- 124.5 nmol/g vs. females 22.8 +/- 16.0 nmol/g, P = 0.048) and caecum (47.6 +/- 31.6 nmol/g vs. females 14.5 +/- 6.6 nmol/g, P = 0.001). Presence of adenomas in the gut influences the intestinal lignan metabolism. Min mice had less intestinal END and ENL as compared with the wild-type mice (P < 0.05). Mean plasma ENL increased 7-fold during the flaxseed feeding (7 nmol/L in control vs. 50 nmol/L in flaxseed group) but no differences between gender and genotype were found. The plasma ENL level did not correlate with adenoma number in the small intestine and colon. CONCLUSION: The number of intestinal adenomas in the Min mouse model is not related to ENL level in plasma nor is it associated with the levels of intestinal lignans. A gender difference in ENL lignan metabolism was found in the gut but not in the plasma.

Consumption of Lactobacillus acidophilus and Bifidobacterium longum do not alter urinary equol excretion and plasma reproductive hormones in premenopausal women.

OBJECTIVE: To confirm the results of an earlier study showing premenopausal equol excretors to have hormone profiles associated with reduced breast cancer risk, and to investigate whether equol excretion status and plasma hormone concentrations can be influenced by consumption of probiotics. DESIGN: A randomized, single-blinded, placebo-controlled, parallel-arm trial. SUBJECTS: In all, 34 of the initially enrolled 37 subjects completed all requirements. INTERVENTION: All subjects were followed for two full menstrual cycles and the first seven days of a third cycle. During menstrual cycle 1, plasma concentrations of estradiol (E(2)), estrone (E(1)), estrone-sulfate (E(1)-S), testosterone (T), androstenedione (A), dehydroepiandrosterone-sulfate (DHEA-S), and sex-hormone-binding globulin (SHBG) were measured on cycle day 2, 3, or 4, and urinary equol measured on day 7 after a 4-day soy challenge. Subjects then received either probiotic capsules (containing Lactobacillus acidophilus and Bifidobacterium longum) or placebo capsules through day 7 of menstrual cycle 3, at which time both the plasma hormone concentrations and the post-soy challenge urinary equol measurements were repeated. RESULTS: During menstrual cycle 1, equol excretors and non-excretors were not significantly different with respect to subject characteristics, diet, or hormone concentrations. Significant inverse correlations were found between E(2) and body mass index (BMI) (P=0.02), SHBG and BMI (P=0.01), DHEA-S and dietary fiber (P=0.04), and A and protein:carbohydrate ratio (P=0.02). Probiotic consumption failed to significantly alter equol excretor status or hormone concentrations during menstrual cycle 3, although there were trends towards decreased concentrations of T (P=0.14) and SHBG (P=0.10) in the probiotic group. CONCLUSIONS: We were unable to verify a previously reported finding of premenopausal equol excretors having plasma hormone concentrations different from those of nonexcretors. Furthermore, a 2-month intervention with probiotic capsules did not significantly alter equol excretion or plasma hormone concentrations.

Highly elevated serum phyto-oestrogen concentrations in patients with diabetic nephropathy.

BACKGROUND AND OBJECTIVES: Phyto-oestrogens, naturally occurring phenolic, hormone-like compounds, have raised considerable interest due to their anticarcinogenic, antiatherogenic and antioxidative potential. Oxidative stress may be one of the key factors in the development of vascular complications in patients with type 1 diabetes. Here, we tested the hypothesis that high concentrations of phyto-oestrogens in serum may be associated with lower occurrence of vascular complications in these patients. SUBJECTS: A total of 400 patients, recruited consecutively from the participant register of the nationwide FinnDiane study of type 1 diabetes and divided into four parallel groups according to the severity of their renal disease with 100 patients to each group: (i) normoalbuminuric patients, (ii) microalbuminuric patients, (iii) macroalbuminuric patients, and (iv) patients with end-stage renal disease (ESRD). MAIN OUTCOME MEASURES: Phyto-oestrogen concentrations in serum (enterolactone, daidzein, genistein and equol) and urine (enterolactone), assessed by time-resolved fluoroimmunoassay. RESULTS: Highly elevated serum concentrations of phyto-oestrogens were measured amongst patients with diabetic nephropathy, and low concentrations amongst patients without diabetic complications. The pattern was similar for all phyto-oestrogens measured, although the increase in mean serum concentrations along with the increasing severity of renal disease was steepest for enterolactone, ranging from 13 nmol L(-1) amongst women and 18 nmol L(-1) amongst men in normoalbuminuric patients to 181 and 206 nmol L(-1) in women and men, respectively, in patients with ESRD (P < 0.001 for both genders between the groups). A strong correlation between serum enterolactone and creatinine concentration was found (r = 0.60, P < 0.001). CONCLUSIONS: The serum concentration of phyto-oestrogens and the severity of diabetic renal disease showed a close positive association, suggesting that phyto-oestrogens are unable to provide any major protective effect, through antioxidative or other mechanisms, on the development of diabetic renal and cardiovascular complications.

Plasma enterolactone or intestinal Bifidobacterium levels do not explain adenoma formation in multiple intestinal neoplasia (Min) mice fed with two different types of rye-bran fractions.

The study was designed to evaluate whether two types of rye-bran fractions result in distinct bifidogenic effect or enterolactone production in multiple intestinal neoplasia (Min) mice and whether these parameters are associated with intestinal tumorigenesis in this animal model. The experimental diets were a non-fibre diet (control), a rye-bran diet, and diets containing either the soluble extract or the insoluble fraction prepared from rye bran. The main result on adenoma formation in these experiments was the observation that the soluble extract increased number (P=0.012) and size (P=0.008) of adenomas in the distal small intestine when compared with the non-fibre group. All rye-supplemented diets supported similarly the in vivo growth of Bifidobacterium (10(8)-10(9) colony forming units/g) in Min mice, whereas the non-fibre diet lowered intestinal Bifidobacterium below the level of detection. The results show that water solubility does not affect the bifidogenicity of rye bran. Mean plasma enterolactone concentration was highest in the rye-bran group (30.0 nmol/l; P=0.002), which along with the soluble-extract group (16.2 nmol/l; P=0.024) differed significantly from the non-fibre diet group (7.5 nmol/l). Thus, the mice fed with the rye bran were the best enterolactone producers. In conclusion, rye bran and rye fractions influence adenoma formation in Min mice to a varying degree but plasma enterolactone levels or the production of bifidogenic bacteria do not mediate the effect.

The effect of flaxseed supplementation in processed foods on serum fatty acids and enterolactone.

OBJECTIVE: The purpose was to study the effects of flaxseed supplementation as a part of daily diet on serum lipids, fatty acids and plasma enterolactone. DESIGN: Eighty volunteers participated in this clinical nutrition study which was carried out in a controlled, double-blind and cross-over manner. The subjects were randomized to diet sequences AB or BA. Diet A meals contained 1.3 g/100 g ground flaxseed and 5 g/100 g flaxseed oil. Also 3-4 g/100 of inulin and wheat fiber was added. AB diet with non-supplemented foods served as control. Test subjects were on both diets for 4 weeks separated by a 4-week wash-out period. Fifteen test subjects continued an open part of the study for 4 additional months. INTERVENTIONS: The dietary intake, basic blood values, serum lipids, fatty acids and enterolactone were measured at baseline, after both intervention periods and during the open study, at baseline and after 2 and 4 months. Serum thiocyanate and blood cadmium were controlled after both intervention periods. RESULTS: The percentage of flaxseed supplemented test food out of total dietary intake was 20% of energy. The test food contained significantly higher amounts of fiber, polyunsaturated fatty acids (PUFAs) and especially alpha-linolenic acid than the control food. No significant changes were observed in the basic laboratory values or in blood lipids. There was a significant increase in serum alpha-linolenic acid, eicosapentaenoic acid and docosapentaenoic acid. Serum enterolactone concentration was doubled during flaxseed supplementation. Serum thiocyanate and blood cadmium values did not exceed reference values and there was no difference between the diets. CONCLUSIONS: In this study we were able to show that, by adding ground flaxseed and flaxseed oil to one or two daily meals, it is possible to obtain significant effects on serum levels of enterolactone and alpha-linolenic acid. SPONSORSHIP: The study was sponsored by the National Technology Agency of Finland (Tekes).

Isoflavone content of the soy based supplements.

A large number of soy isoflavone products with indications of possible health effects are available on the market. Fifteen different soy based products were analyzed using high performance liquid chromatography (HPLC) with coulometric electrode array detector to determine the total amount of isoflavones in aglycones after the hydrolysis and identify the different forms of the isoflavone conjugates. The aim of the study was to evaluate how well the isoflavone content data supplied by the producers correspond to our analysis results. Only one product contained isoflavones measured in aglycones the same amount as was the value given by the producer. The total amount of the isoflavones in aglycones ranged from 0.121 to 201 mg/g. Measured amounts of isoflavones in aglycones after the hydrolysis were in general lower than the values in the product labels. Product data were often confusing and the concrete amount of isoflavones was difficult to find out.

Dietary soy phytoestrogen effects on brain structure and aromatase in Long-Evans rats.

Phytoestrogens are estrogen-like (plant-derived) molecules that protect against age-related diseases (cardiovascular disease and osteoporosis), hormone-dependent (breast and prostate) cancers and selectively bind estrogen receptors. However, little is known about the influence of phytoestrogens on brain. Using diets containing either high phytoestrogen levels, derived from soy, or very low phytoestrogens we quantified phytoestrogen concentrations of daidzein, genistein and equol in brain. We found that dietary phytoestrogens: significantly decrease body and prostate weights, do not alter brain aromatase levels and significantly change during adulthood the structure of the sexually dimorphic brain region (i.e. anteroventral periventricular nucleus; AVPV) in male, but not in female rats. Since most commercial animal diets contain significant concentrations of phytoestrogens their influence on brain structure should be considered.

In vitro metabolism of plant lignans: new precursors of mammalian lignans enterolactone and enterodiol.

The metabolism of the plant lignans matairesinol, secoisolariciresinol, pinoresinol, syringaresinol, arctigenin, 7-hydroxymatairesinol, isolariciresinol, and lariciresinol by human fecal microflora was investigated to study their properties as mammalian lignan precursors. The quantitative analyses of lignan precursors and the mammalian lignans enterolactone and enterodiol were performed by HPLC with coulometric electrode array detector. The metabolic products, including mammalian lignans, were characterized as trimethylsilyl derivatives by gas chromatography-mass spectrometry. Matairesinol, secoisolariciresinol, lariciresinol, and pinoresinol were converted to mammalian lignans only. Several metabolites were isolated and tentatively identified as for syringaresinol and arctigenin in addition to the mammalian lignans. Metabolites of 7-hydroxymatairesinol were characterized as enterolactone and 7-hydroxyenterolactone by comparison with authentic reference compounds. A metabolic scheme describing the conversion of the most abundant new mammalian lignan precursors, pinoresinol and lariciresinol, is presented.

Intake of dietary phytoestrogens is low in postmenopausal women in the United States: the Framingham study(1-4).

Many plants that are consumed contain phytoestrogens. Only a few published studies have examined the dietary intake of phytoestrogens in the general Western population. The potentially positive health effects of phytoestrogens might be of relevance to postmenopausal women. The aim of the present study was to estimate the intake of dietary isoflavones, coumestans and lignans by healthy Western postmenopausal women. For this purpose, we studied 964 postmenopausal, Caucasian women who participated in the Framingham Offspring Study and completed the Willett food-frequency questionnaire (FFQ). By searching the medical and agricultural literature and contacting experts, we identified food sources of phytoestrogens. The concentrations of the different isoflavones, coumestrol and lignans in each food in the FFQ were scored in seven categories and multiplied by the serving size of the food and the frequency of its consumption. The estimated daily median intake of the isoflavone daidzein was 39 microg (24-57 microg); of genistein, 70 microg (28-120 microg); of formononetin, 31 microg (13-44 microg); and of biochanin A, 6 microg (2-11 microg). Median total intake of isoflavones was 154 microg (99-235 microg). The main sources of isoflavones were beans and peas. The estimated daily intake of coumestans was 0.6 microg (0.2-1.7 microg), with broccoli as the main source. The estimated daily median intake of matairesinol was 19 microg (12-28 microg) and of secoisolariciresinol 560 microg (399-778 microg). The median total intake of lignans was 578 microg (416-796 microg). The main source of the lignans was fruits. The daily dietary intake of phytoestrogens in healthy postmenopausal Caucasian women in the United States is <1 mg.

Urinary phytoestrogens and postmenopausal breast cancer risk.

Phytoestrogens are defined as plant substances that are structurally or functionally similar to estradiol. We report the associations of two major phytoestrogens, genistein and enterolactone, with breast cancer risk, using urinary specimens collected 1-9 years before breast cancer was diagnosed. The subjects were 88 breast cancer cases and 268 controls, selected from a cohort of postmenopausal women (n = 14,697) who participated in a breast cancer screening program. Mean levels of urinary genistein and enterolactone were determined by time resolved fluoroimmunoassay, using an average of two overnight urinary samples obtained from each participant on the first and the second screening rounds with a time interval of approximately 1 year. Odds ratios (ORs) of the highest to the lowest tertile of urinary phytoestrogen/creatinine concentrations and 95% confidence intervals (CIs) were computed. Higher urinary genistein excretion was weakly and nonsignificantly associated with a reduced breast cancer risk. OR for the highest tertile compared with lowest tertile was 0.83; 95% CI, 0.46-1.51. Higher urinary enterolactone excretion was weakly and nonsignificantly associated with an increased breast cancer risk. OR for the highest tertile compared with the lowest tertile was 1.43; 95% CI, 0.79-2.59. Tests for trends for both phytoestrogens were nonsignificant. We were not able to detect the previously reported protective effects of genistein and enterolactone on breast cancer risk in our postmenopausal population of Dutch women. Such an effect may be smaller than expected and/or limited to specific subgroups of the population.

Visual spatial memory is enhanced in female rats (but inhibited in males) by dietary soy phytoestrogens.

BACKGROUND: In learning and memory tasks, requiring visual spatial memory (VSM), males exhibit superior performance to females (a difference attributed to the hormonal influence of estrogen). This study examined the influence of phytoestrogens (estrogen-like plant compounds) on VSM, utilizing radial arm-maze methods to examine varying aspects of memory. Additionally, brain phytoestrogen, calbindin (CALB), and cyclooxygenase-2 (COX-2) levels were determined. RESULTS: Female rats receiving lifelong exposure to a high-phytoestrogen containing diet (Phyto-600) acquired the maze faster than females fed a phytoestrogen-free diet (Phyto-free); in males the opposite diet effect was identified. In a separate experiment, at 80 days-of-age, animals fed the Phyto-600 diet lifelong either remained on the Phyto-600 or were changed to the Phyto-free diet until 120 days-of-age. Following the diet change Phyto-600 females outperformed females switched to the Phyto-free diet, while in males the opposite diet effect was identified.Furthermore, males fed the Phyto-600 diet had significantly higher phytoestrogen concentrations in a number of brain regions (frontal cortex, amygdala & cerebellum); in frontal cortex, expression of CALB (a neuroprotective calcium-binding protein) decreased while COX-2 (an inducible inflammatory factor prevalent in Alzheimer's disease) increased. CONCLUSIONS: Results suggest that dietary phytoestrogens significantly sex-reversed the normal sexually dimorphic expression of VSM. Specifically, in tasks requiring the use of reference, but not working, memory, VSM was enhanced in females fed the Phyto-600 diet, whereas, in males VSM was inhibited by the same diet. These findings suggest that dietary soy derived phytoestrogens can influence learning and memory and alter the expression of proteins involved in neural protection and inflammation in rats.

Isoflavone phytoestrogens consumed in soy decrease F(2)-isoprostane concentrations and increase resistance of low-density lipoprotein to oxidation in humans.

BACKGROUND: Oxidative damage to lipids may be involved in the etiology of atherosclerosis, cardiovascular disease in general, and cancer. The soy isoflavone phytoestrogens, genistein and daidzein, and equol (a daidzein metabolite produced by intestinal microflora) are antioxidants in vitro; equol is a particularly good inhibitor of LDL oxidation and membrane lipid peroxidation. OBJECTIVE: We sought to investigate the effects of a diet enriched with soy containing isoflavones on in vivo biomarkers of lipid peroxidation and resistance of LDL to oxidation, compared with a diet enriched with soy from which the isoflavones had been extracted. DESIGN: : A randomized, crossover design was used to compare diets enriched with soy that was low or high in isoflavones in 24 subjects. Plasma concentrations of an F(2)-isoprostane, 8-epi-prostaglandin F(2)(alpha) (8-epi-PGF(2)(alpha)), a biomarker of in vivo lipid peroxidation, and resistance of LDL to copper-ion-induced oxidation were determined. RESULTS: Plasma concentrations of 8-epi-PGF(2)(alpha) were significantly lower after the high-isoflavone dietary treatment than after the low-isoflavone dietary treatment (326 +/- 32 and 405 +/- 50 ng/L, respectively; P = 0.028) and the lag time for copper-ion-induced LDL oxidation was longer (48 +/- 2.4 and 44 +/- 1.9 min, respectively; P = 0.017). Lag time for oxidation of unfractionated plasma and plasma concentrations of malondialdehyde, LDL alpha-tocopherol, polyunsaturated fatty acids, and isoflavonoids did not differ significantly between dietary treatments. CONCLUSIONS: Consumption of soy containing naturally occurring amounts of isoflavone phytoestrogens reduced lipid peroxidation in vivo and increased the resistance of LDL to oxidation. This antioxidant action may be significant with regard to risk of atherosclerosis, cardiovascular disease in general, and cancer.

Rapid analysis of phytoestrogens in human urine by time-resolved fluoroimmunoassay.

A time-resolved fluoroimmunoassay (TR-FIA), with europium labeled phytoestrogens as tracers, was developed for the quantitative determination of enterolactone, genistein and daidzein in human urine. The aim was to create a method for the screening of large populations in order to assess the possible correlations between the urinary levels and the risk of Western diseases. After the synthesis of the 5'-carboxymethoxy derivative of enterolactone and 4'-O-carboxymethyl derivatives of daidzein and genistein, the respective compound was coupled to bovine serum albumin and then used as an antigen in the immunization of rabbits. The same derivatives of the phytoestrogen were used in preparing the europium tracers. After the enzymatic hydrolysis, the TR-FIA was carried out using the Victor 1420 multilabel counter. The method has sufficient sensitivity to measure the phytoestrogens at concentrations even below 5 nmol/l. The intra- and inter-assay coefficients of variation, at three different concentrations, varied from 1.9 to 5.3 and from 2.4 to 9.7, respectively. We measured urinary enterolactone, genistein and daidzein in 215 samples from Finnish healthy women and found that more than 50% of the values ranged between 1 and 7, <0.1 and 0.6 and below 0.6 micromol/24 h, respectively. The TR-FIA method including only a hydrolysis step gave higher values than those measured by gas chromatography-mass spectrometry (GC-MS). However, the assay results by the present method showed strong correlation with those obtained by GC-MS. It is concluded that the TR-FIA is suitable for population screening of urinary phytoestrogens.

Dietary soy-derived isoflavone phytoestrogens. Could they have a role in coronary heart disease prevention?

Soy protein-containing foods are a rich source of isoflavone phytoestrogens, such as genistein and daidzein. There is great interest in these substances, as lower rates of chronic diseases, including coronary heart disease, have been associated with high dietary intake of soy-containing foods. Soy phytoestrogens bind weakly to estrogen receptors, and some bind more strongly to estrogen receptor-beta compared with estrogen receptor-alpha. A meta-analysis has indicated that isoflavone phytoestrogens lowered plasma cholesterol concentrations in subjects with initially elevated levels, but had little effect in subjects with normal cholesterol concentrations. These substances reportedly may also have beneficial effects on arterial endothelial function. In addition to these potentially antiatherogenic effects, many laboratories are investigating other possible mechanisms, including antioxidative and antiproliferative properties of these substances. We have shown that dietary supplementation with soy-derived isoflavones reduced the in vitro oxidation susceptibility of low-density lipoprotein (LDL). To further explore this phenomenon, we incorporated genistein and daidzein into LDL molecules in vitro with the aid of an artificial transfer system. However, it was necessary to convert the isoflavone molecules to fat-soluble derivatives, fatty acid esters (analogous to esterified endogenous estrogens, which are known to occur in vivo), to achieve significant incorporation. The LDLs containing esterified isoflavones were shown to be less susceptible to oxidation in vitro than native LDL. We also employed U937 cell cultures for investigating the effects of isoflavone-containing LDLs on cell proliferation. Some of these LDLs exhibited antiproliferative effects in cultured U937 cells. In summary, lipophilic phytoestrogen derivatives could be incorporated into LDLs, increasing their oxidation resistance and antiproliferative efficacy ex vivo, both of which are, in theory, antiatherogenic effects. Further studies are needed to assess to what extent analogous effects could be produced in vivo and whether such substances have a role in hormone replacement and coronary heart disease prevention in postmenopausal women.