K. Wähälä

Determination of urinary lignans and phytoestrogen metabolites, potential antiestrogens and anticarcinogens, in urine of women on various habitual diets

Recently two groups of compounds with diphenolic structure, the lignans and the isoflavonic phytoestrogens, were detected and identified in human urine and other biological fluids. These compounds are of great biological interest because they exhibit both in vitro and in vivo weak estrogenic and sometimes also antiestrogenic activities and many plant lignans have been shown to have anticarcinogenic, antiviral, antifungal and other interesting biological effects. The compounds found in relatively large amounts (10-1000 times more than estrogens) in urine are modified by intestinal bacteria from plant lignans and phytoestrogens, which are present in fiber-rich food such as grain and beans. They bind with low affinity to estrogen receptors and preliminary results suggest that they may induce production of sex hormone binding globulin (SHBG) in the liver and in this way may influence sex hormone metabolism and biological effects. Five compounds, the lignans enterolactone (Enl), enterodiol (End) and the isoflavonic phytoestrogen metabolites daidzein (Da), equol (Eq) and O-desmethylangolensin (O-Dma) were measured in urine by gas chromatography-mass spectrometry (selected ion monitoring) using deuterated internal standards in 5 groups of women (total number 53). The members of three dietary groups (omnivores, lactovegetarians and macrobiotics) were living in Boston and of two groups in Helsinki (omnivores and lactovegetarians). Until now measurements have been carried out in 94 72-h samples. The highest mean excretion of the most abundant compound, enterolactone, was found in the macrobiotic group and the lowest in the omnivoric groups. Total mean 24-h excretion of enterolactone was 17,680 nmol in the macrobiotics, 4,170 nmol in the Boston lactovegetarians, 3,650 nmol in the Helsinki lactovegetarians, 2,460 nmol in the Helsinki omnivores and 2,050 nmol in the Boston omnivores. The other diphenols followed approximately the same pattern. In an earlier study the lowest excretion of enterolactone (1,040 nmol/24 h) was found in a group of postmenopausal apparently healthy breast cancer patients living in Boston. It is concluded that further studies are necessary to elucidate the possible role of these compounds in cancer and other diseases. However, the evidence obtained until now seems to justify the conclusion that these compounds may be among the dietary factors affording protection against hormone-dependent cancers in vegetarians and semivegetarians.

Lignan and isoflavonoid concentrations in tea and coffee.

Tea is a beverage consumed widely throughout the world. The existence in tea of chemopreventing compounds possessing antimutagenic, anticarcinogenic and antioxidative properties has been reported. High intakes of tea and foods containing flavonoids have recently been shown to be negatively correlated to the occurrence of CHD. However, tea may contain other compounds with similar activities. Using a new gas chromatographic-mass spectrometric method we measured lignans and isoflavonoids in samples of twenty commercial teas (black, green and red varieties) and, for comparison, six coffees. Both unbrewed and brewed tea were investigated. The analysis of the teas yielded relatively high levels of the lignans secoisolariciresinol (5.6-28.9 mg/kg; 15.9-81.9 mumol/kg) and matairesinol (0.56-4.13 mg/kg; 1.6-11.5 mumol/kg) but only low levels of isoflavonoids. Because the plant lignans, as well as their mammalian metabolites enterolactone and enterodiol, have antioxidative properties and these mammalian lignans occur in high concentrations in plasma, we hypothesize that lignan polyphenols may contribute to the protective effect of tea on CHD.

Phyto-oestrogen content of berries, and plasma concentrations and urinary excretion of enterolactone after a single strawberry-meal in human subjects

Quantitative data on phyto-oestrogen, particularly lignan, content in edible plants are insufficient. We, therefore, measured isoflavonoids and lignans in nine edible berries using an isotope dilution gas chromatography-mass spectrometry method for foods and found substantial concentrations of the lignan secoisolariciresinol (1.39-37.18 mg/kg DM), low amounts of matairesinol (0-0.78 mg/kg DM) and no isoflavones. To determine pharmacokinetics and urinary excretion pattern of the mammalian lignan enterolactone derived from plant lignans, a study with human subjects was conducted. Five healthy women and two men consumed, after a 72 h period of a phyto-oestrogen-free regimen, a single strawberry-meal containing known amounts of plant lignans. Basal and post-meal blood and urine samples were collected at short intervals. The samples were analysed using time-resolved fluoroimmunoassay of enterolactone. The meal increased plasma concentration of enterolactone after 8-24 h and in urine in the 13-24 h and 25-36 h urine collections. High individual variability of the metabolic response was observed. Enterolactone excreted in the urine collected throughout the 48 h post-meal yielded on average 114% of the plant lignans consumed. It is concluded that berries containing relatively high concentrations of plant lignans contribute to plasma and urinary levels of mammalian enterolactone in human subjects.

Inhibitory effects of soy and rye diets on the development of Dunning R3327 prostate adenocarcinoma in rats

Dunning R3327 PAP prostate tumors were transplanted in 125 rats, the rats were divided into five groups, and tumor development was examined for 24 weeks during treatment with diets containing 33% of soy flour (SD), rye bran (RB), heat‐treated rye bran (HRB), or rye endosperm (RE).

Identification in human urine of a natural growth inhibitor for cells derived from solid paediatric tumours

Abstract. Partially purified urine of healthy human subjects contains several fractions able to inhibit the proliferation of cultured human neuroblastoma cells. One of the most active fractions was further analysed by gas chromatography‐mass spectrometry and shown to contain genistein, a substance formed in the human body from precursors obtained by diet. Synthetic genistein was able to inhibit the proliferation of human neuroblastoma cells with a half‐maximal effect at 5–10 μmol 1‐1 concentrations. Genistein displayed similar potencies in inhibiting the proliferation of cells derived from various other solid pediatric tumours. Our results suggest that genistein is a natural antineo‐plastic agent present in diet and that it could be useful for the therapy of paediatric tumours.

Detection and identification of the plant lignans lariciresinol, isolariciresinol and secoisolariciresinol in human urine

The mammalian lignans enterolactone and enterodiol are regular constituents of human urine and are excreted daily in mumol amounts. They are produced by metabolic action of intestinal bacteria from natural plant lignan precursors which are constituents of various food plants. However, natural plant lignans seem to occur very seldom in detectable amounts in human urine. The present investigation shows that only in 5% of the 150 diphenolic fractions extracted from the urine of women plant lignans other than the previously identified matairesinol could be found. The lignans lariciresinol, isolariciresinol and secoisolariciresinol were identified for the first time by comparison of their GC characteristics and mass spectra with the corresponding results of authentic synthesized reference compounds. Secoisolariciresinol is one natural precursor of the mammalian lignan enterodiol. Of the two other plant lignans, no animal or human metabolic products are known. The occurrence of chemically unchanged plant lignans in some cases in human urine could be a result of an insufficient metabolic capacity of intestinal bacteria, resulting in a decreased detoxification of phenolic plant products.

Potentially hallucinogenic 5-hydroxytryptamine receptor ligands bufotenine and dimethyltryptamine in blood and tissues

Bufotenine and N,N‐dimethyltryptamine (DMT) are hallucinogenic dimethylated indolethylamines (DMIAs) formed from serotonin and tryptamine by the enzyme indolethylamine N‐methyltransferase (INMT) ubiquitously present in non‐neural tissues. In mammals, endogenous bufotenine and DMT have been identified only in human urine. The DMIAs bind effectively to 5HT receptors and their administration causes a variety of autonomic effects, which may reflect their actual physiological function. Endogenous levels of bufotenine and DMT in blood and a number of animal and human tissues were determined using highly sensitive and specific quantitative mass spectrometric techniques. A new finding was the detection of large amounts of bufotenine in stools, which may be an indication of its role in intestinal function. It is suggested that fecal and urinary bufotenine originate from epithelial cells of the intestine and the kidney, respectively, although the possibility of their synthesis by intestinal bacteria cannot be excluded. Only small amounts of the DMIAs were found in somatic or neural tissues and none in blood. This can be explained by rapid catabolism of the DMIAs by mitochondrial monoamino‐oxidase or by the fact that the dimethylated products of serotonin and tryptamine are not formed in significant amounts in most mammalian tissues despite the widespread presence of INMT in tissues.

Effect of Dietary Flaxseed on Serum Levels of Estrogens and Androgens in Postmenopausal Women

Flaxseed is a rich source of dietary lignans. Experimental studies suggest lignans may exert breast cancer preventive effects through hormonal mechanisms. Our aim was to study the effects of flaxseed on serum sex hormones implicated in the development of breast cancer. Forty-eight postmenopausal women participated in a 12-wk preintervention–postintervention study. Participants consumed 7.5 g/day of ground flaxseed for the first 6 wk and 15.0 grams/day for an additional 6 wk. Nonsignificant declines were noted over the 12 wk (95% confidence intervals) for estradiol (pg/ml), estrone (pg/ml), and testosterone (pg/ml): –4.4 (–12.6 to 3.9), –3.3 (–7.7 to 1.2), –4.7 (–17.8 to 8.5), respectively. Changes tended to be more pronounced in overweight/obese women, particularly for estrone (–6.5, –11.9 to –1.2; P = .02). Our results suggest that dietary flaxseed may modestly lower serum levels of sex steroid hormones, especially in overweight/obese women.

Effect of Flaxseed Consumption on Urinary Levels of Estrogen Metabolites in Postmenopausal Women

Flaxseed is a rich source of dietary lignans. It has been hypothesized that lignans may decrease breast cancer risk through modulation of endogenous hormone levels. The aim of this study was to determine the effect of flaxseed supplementation on urinary levels of estrogen metabolites that may be involved in the development of breast cancer. Forty-three postmenopausal women participated in this 12-wk preintervention–postintervention study. Participants consumed 7.5 g/day of ground flaxseed for 6 wk, followed by 15 g/day for an additional 6 wk. The mean urinary level of 16α –hydroxyestrone (16α -OHE1) was higher at the end of 12 wk compared to baseline (change of 1.32 ug/day, P = 0.02). There was no significant change in 2-OHE1 excretion. The mean urinary level of the 2-OHE1/16α -OHE1 ratio was lower at the end of 12 wk compared to baseline (change of −1.1, P = 0.02). Mean urinary excretion of 2-methoxyestradiol was also lower at 12 wk than at baseline ( P = 0.03). Based on the current paradigm of the effects of estrogen metabolism on breast cancer risk, the regimen of dietary flaxseed intake used in this study did not appear to favorably alter breast cancer risk through shifts in estrogen metabolism pathways in postmenopausal women.

Immunoassay of Phytoestrogens in Human Plasma

Highly sensitive plasma immunoassay methods based on time-resolved fluorometry were developed for plasma enterolactone, genistein, and daidzein. For daidzein and genistein three types of methods and for enterolactone two different methods were developed and validated and the results compared with our gas chromatographic-mass spectrometric reference method. All three compounds may be determined in duplicate in a 2-300 mu plasma sample, even in subjects with low phytoestrogen values.