Analysis of serum bile acids by isotope dilution-mass spectrometry to assess the performance of routine total bile acid methods.

BACKGROUND: WEQAS, one of the largest EQA (External Quality Assessment) providers in the UK, offers a bile acid EQA scheme, with linear serum pools containing cholic acid, deoxycholic acid and chenodeoxycholic acid, reflecting levels observed in obstructive cholestasis. Total bile acids are currently measured routinely by non-specific enzymatic methods. Traceability of results to the SI unit utilizing reference target values is the preferred method of comparison of returned results where available, ensuring the transfer of accuracy from definitive methods to routine methods. METHODS: Target values have been assigned to EQA material utilizing isotope dilution gas chromatography mass spectrometry (ID-GCMS). The methodology was based on published routine methods and adapted for use as a ID-GCMS target method. The total bile acid target value was reported as the sum of the three major bile acids measured: cholic acid, deoxycholic acid and chenodeoxycholic acid. RESULTS: The produced target values have been used to assess the performance of total bile acid methods. A degree of variability was observed between the third-generation enzyme-formazan methods and the fifth-generation thio-NADH methods. Additionally, the Sentinel results showed a positive bias in comparison to their peer formazan method group. CONCLUSION: The use of ID-GCMS target results provides a common comparison for resumed results in EQA schemes, highlighting any method differences. Thus can then aid in the harmonisation of results observed for each of the different methods.

Dietary flour supplementation decreases post-menopausal hot flushes: effect of soy and wheat.

Plants contain compounds with oestrogen--like action called phytoestrogens. Soy contains daidzin, a potent phytoestrogen, and wheat flour contains less potent enterolactones. We aimed to show in 58 postmenopausal women (age 54, range 30-70 years) with at least 14 hot flushes per week, that their daily diet supplemented with soy flour (n = 28) could reduce flushes compared with wheat flour (n = 30) over 12 weeks when randomised and double blind. Hot flushes significantly decreased in the soy and wheat flour groups (40% and 25% reduction, respectively < 0.001 for both) with a significant rapid response in the soy flour group in 6 weeks (P < 0.001) that continued. Menopausal symptom score decreased significantly in both groups (P < 0.05). Urinary daidzein excretion confirmed compliance. Vaginal cell maturation, plasma lipids and urinary calcium remained unchanged. Serum FSH decreased and urinary hydroxyproline increased in the wheat flour group.

Phyto-oestrogens and risk of prostate cancer in Scottish men.

A population-based case-control study of diet, inherited susceptibility and prostate cancer was undertaken in the lowlands and central belt of Scotland to investigate the effect of phyto-oestrogen intake and serum concentrations on prostate cancer risk. A total of 433 cases and 483 controls aged 50-74 years were asked to complete a validated FFQ and provide a non-fasting blood sample. Multivariate logistic regression analysis found significant inverse associations with increased serum concentrations of enterolactone (adjusted OR 0.40, 95 % CI 0.22, 0.71] and with the consumption of soy foods (adjusted OR 0.52, 95 % CI 0.30, 0.91). However, no significant associations were observed for isoflavone intake or serum genistein, daidzein and equol. This study supports the hypotheses that soy foods and enterolactone metabolised from dietary lignans protect against prostate cancer in older Scottish men.

Phyto-oestrogen intake in Scottish men: use of serum to validate a self-administered food-frequency questionnaire in older men.

OBJECTIVE: To study dietary intake and serum concentrations of isoflavones in order to provide relative validation of isoflavone intake estimates from the Scottish Collaborative Group - Food-Frequency Questionnaire (SCG-FFQ). DESIGN: Validation study. SETTING: Southern Scotland. METHOD: Dietary intake of isoflavones was estimated using the semiquantitative SCG-FFQ and rank correlation and Kappa statistics were used for the relative validation of intakes against serum isoflavone concentrations in 203 male participants who were population controls in a case-control study of diet and prostate cancer. RESULTS: The median intake of isoflavones (daidzein and genistein) was 1.0mg/day (l-QR 0.6-1.8). The median serum concentration of genistein was 33.79 nmol/l (I-QR 14.12-64.93), nearly twice that of daidzein (18.00 nmol/l, I-QR 8.26-29.45). Equol was detected in 49% of subjects; in these subjects the median was 0.67 nmol/l (I-QR 0.34-1.51). Isoflavone intake was significantly correlated with serum concentrations of daidzein (p = 0.24, P = 0.001), genistein (p = 0.26, P < 0.001) and total isoflavonoids (sum of daidzein, genistein and equol) ( p = 0.27, P < 0.001). Whereas values for weighted Kappa ranged from 0.16 (P = 0.002) for daidzein and equol combined to 0.22 (P < 0.001) for genistein. CONCLUSIONS: These results demonstrate the suitability of the SCG-FFQ to rank usual isoflavone intakes in older Scottish men, a population observed to have low consumption of soy foods.

Investigation of the reliability of 24 h urine excretion as a biomarker of isoflavone exposure over time and over a wide range of isoflavone intakes.

OBJECTIVE: To study the variation in genistein + daidzein intake over a 6-month period and test the reliability of 24 h urinary isoflavones as a biomarker of exposure over time. DESIGN: Dietary genistein + daidzein intake was assessed at various time points throughout six months in 15 healthy subjects. Group 1 (n=8) followed nonsupplemented diets and Group 2 (n=7) took a 35 mg/d isoflavone supplement for 3 months and each subject provided a 24 h urine collection, validated with para-aminobenzoic acid, during weeks 7, 15 and 19. Urine was analysed for genistein and daidzein using LC-MS. RESULTS: Isoflavone intake in Groups 1 and 2 ranged from 0.00 to 1.1 mg/d and 0.1 to 53.1 mg/d, respectively. Urine excretion for both groups ranged from 0.20 to 9.56 mg/d. The relationship between 24 h excretion and isoflavone intake is y=0.44 x +/- 0.03(standard deviation) + 1.57; r=0.89, P<0.001. CONCLUSION: The 24 h urinary isoflavones can be used as biomarkers of isoflavone exposure over time.

Effect of red clover-derived isoflavone supplementation on insulin-like growth factor, lipid and antioxidant status in healthy female volunteers: a pilot study.

BACKGROUND: Isoflavones are estrogen-like plant compounds that may protect against cardiovascular disease and endocrine-responsive cancer. Isoflavones may, because of their ability to act as selective estrogen receptor modulators, alter insulin-like growth factor (IGF) status. OBJECTIVE: The aim of this study was to assess the effect of 1-month isoflavone supplementation (86 mg/day red clover-derived isoflavones) on IGF status. DESIGN AND SUBJECTS: Healthy pre- (n=16) and postmenopausal (n=7) women were invited to take part in a randomised, placebo-controlled crossover study with a minimum 2-month washout period. RESULTS: : For premenopausal subjects, the change in IGF-1, IGF-BP1 and IGF-BP3 assessed at different points of the menstrual cycle did not differ between isoflavone and placebo phase. However, the change in IGF-1, when examined pre- and post-supplementation, was nonsignificantly reduced (P=0.06) on the isoflavone supplement compared to placebo. For postmenopausal subjects, the change in IGF-1, IGF-BP1 and IGFBP-3 concentrations over the supplementation period did not differ between isoflavone or placebo phase. Isoflavones increased HDL in postmenopausal women compared to placebo (P=0.02) but did not alter either cholesterol or triacylglycerol concentrations, and had no effect on antioxidant status. CONCLUSIONS: This study shows that 1-month supplementation with red clover isoflavones has a positive effect on HDL cholesterol, but at most a small effect on IGF status in premenopausal and no effect in postmenopausal subjects. Further studies are required to ascertain the role these dietary compounds may have to play in breast cancer prevention.

Two-week dietary soy supplementation has an estrogenic effect on normal premenopausal breast.

An association has been reported between consumption of a high soy diet and a low incidence of breast cancer within populations of Southeast Asia. Phytoestrogens present in soy act as partial estrogen agonists or antagonists and can inhibit breast cancer cell proliferation in vitro. The effect of 14-day dietary soy supplementation with 60 g (45 mg isoflavones) on the normal breast of 84 premenopausal patients was determined. Serum concentrations of the isoflavanoids, genistein, daidzein, and equol, were raised in patients after soy supplementation (P < or = 0.025). Nipple aspirate (NA) levels of genistein and daidzein were higher than paired serum levels, both before (P < 0.001 and P = 0.001, respectively) and after soy supplementation (P < 0.001 and P = 0.049, respectively); however, there was no significant increase in NA isoflavone levels in response to soy. NA levels of apolipoprotein D were significantly lowered and pS2 levels raised in response to soy supplementation (P < or = 0.002), indicative of an estrogenic stimulus. No effect of soy supplementation on breast epithelial cell proliferation, estrogen and progesterone receptor status, apoptosis, mitosis, or Bcl-2 expression was detected. In conclusion, short term dietary soy has a weak estrogenic response on the breast, as measured by nipple aspirate apolipoprotein D and pS2 expression. No antiestrogenic effect of soy on the breast was detected.

Dietary soy supplementation and phytoestrogen levels.

OBJECTIVE: To investigate the relationship between levels of phytoestrogens in blood and urine and symptom control in postmenopausal women whose diets were supplemented with soy containing high levels of phytoestrogen. METHODS: Phytoestrogen levels in blood and urine were correlated with the number of hot flushes and vaginal maturation indices in 104 postmenopausal women whose diets were supplemented with 60 g of soy powder daily for 3 months in a double-masked, parallel, placebo-controlled trial. The effect of soy supplementation on menopausal symptoms in this study population was reported previously. RESULTS: Serum levels of genistin, daidzin, and equol were significantly higher in women after soy diet supplementation (+756%, +593%, +1008%, and 57% respectively). The concentration of enterolactone in the soy group was increased by 57% compared with baseline, but the difference with placebo was not statistically significant. The increase in phytoestrogen levels in the soy group in blood or urine did not correlate with fewer hot flushes. Vaginal maturation indices did not change in the soy group. CONCLUSION: Phytoestrogen levels increased in women who consumed soy supplement, but that does not fully explain climacteric symptom reduction. It is possible that other types of yet unknown phytoestrogens or components in soy other than phytoestrogen influence hot flushes.

Diet and its preventive role in prostatic disease.

Asian men have much lower incidences of prostate cancer and possibly of benign prostatic hyperplasia (BPH) than their Western counterparts. Vegetarian men also have a lower incidence of prostate cancer than omnivorous males. Both Asian and vegetarian men consume low-fat, high-fibre diets which provide a rich supply of weak dietary oestrogens. These plant or phyto-oestrogens have been proposed as chemopreventive agents, particularly for Asian men and to a lesser extent, for vegetarian men also. The three principal classes of phyto-oestrogens are the isoflavonoids, flavonoids and lignans. Many foods of plant origin contain varying amounts of these compounds and hundreds of plants manifest some degree of oestrogenic activity. Soya, a dietary staple in many parts of Asia, is a major source of the isoflavonoids, daidzein and genistein. Flavonoids are present in high concentration in many fruits, vegetables and crop species. In particular, apigenin and kaempferol are regarded as major flavonoids because of their common occurrence in plants, and their significant concentrations when present. Apples, onions and tea-leaves are excellent sources of flavonoids. Plant lignans are present in many cereals, grains, fruits and vegetables, and give rise to the mammalian lignans, enterodiol and enterolactone; however, the richest source is linseed (flaxseed) and other oilseeds. In addition to their oestrogenic activity, many of these plant compounds can interfere with steroid metabolism and bioavailability, and also inhibit enzymes, such as tyrosine kinase and topoisomerase, which are crucial to cellular proliferation.

Certain aspects of molecular endocrinology that relate to the influence of dietary factors on the pathogenesis of prostate cancer.

Isoflavonoids, flavonoids and lignans are natural oestrogenic compounds derived from soya, tea, fruits and vegetables and they have been proposed as chemopreventive agents in Asian men, in whom the incidence of prostate cancer is much lower than in men from the West. In addition to their weak oestrogenic activity, oestrogen antagonistic activity has also been described for some of these compounds. Furthermore, the lignan, enterolactone and the soya-derived isoflavone genistein are inhibitors of several steroid metabolising enzymes, such as aromatase, 5alpha-reductase and 17beta-hydroxysteroid dehydrogenase. Genistein is a potent inhibitor of tyrosine kinases and along with flavonoids such as kaempferol and apigenin is also an inhibitor of topoisomerases I and II, enzymes which are crucial to cellular proliferation. Genistein is also an inhibitor of angiogenesis and many experimental in vivo and in vitro models, including those for prostate cancer, are growth inhibited by isoflavonoids, flavonoids and lignans. It is estimated that the traditionally eating Japanese male consumes approximately 20 mg of isoflavones per day, whereas for Western men, the daily consumption would be less than 1 mg/day. This is reflected in a high mean plasma concentration of genistein (180 ng/ml, n = 72) in Japanese men, compared to a level of <10 ng/ml for Western males.

Phyto-oestrogen excretion and rate of bone loss in postmenopausal women.

OBJECTIVE: The hypothesis was tested that the rate of postmenopausal bone loss is inversely associated with long-term urinary excretion of phyto-oestrogens, as a marker of habitual dietary intake. DESIGN: Secondary analysis of a 10-year follow-up study (1979 1989) among postmenopausal women in the Netherlands. SUBJECTS: From the original population of 154 women, 32 women were selected with an annual rate of radial bone loss of < or = 0.5% over the first 5 years of the study and 35 women with a rate of > or = 2.5% per year. METHODS: The isoflavonoids genistein, daidzein and equol, and the lignan enterolactone were determined by gas chromatography mass spectrometry in aggregate samples from annually collected urine samples. Cortical bone density of the radius had previously been measured annually by single-photon absorptiometry. RESULTS: Excretion of isoflavonoids did not differ between both groups, although in multivariate analysis equol excretion was weakly positively associated with rate of bone loss in the 5 years after the menopause. Enterolactone excretion was significantly higher in the group with high rate of bone loss. This positive association remained in multivariate linear regression analysis after adjustment for age, years since menopause, body mass index and intake of calcium, vegetable protein and dietary fibre. CONCLUSIONS: Enterolactone excretion is likely to be an indicator of consumption of grains and legumes; it is not clear whether the observed positive association with rate of bone loss is a causal one. Our results do not support a preventive effect of low, unsupplemented dietary intake of phyto-oestrogens on postmenopausal cortical bone loss. However, no conclusions can be drawn about effects of higher doses of phyto-oestrogens.

Phytoestrogens and diseases of the prostate gland.

Both benign hyperplasia (BPH) and cancer of the prostate are manifest in men beyond the age of 50. Approximately 50% of men greater than 50 years of age will suffer from the symptoms associated with BPH, especially from bladder outlet obstruction. With the ever-increasing proportion of the population over 65 years of age worldwide, BPH is becoming an important medical problem as the world moves into the next millennium. Cancer of the prostate is the second most commonly diagnosed cancer after skin cancer in the male population of the United States, and the second most common cause of death from cancer after that of the lung. Overall, around the world the incidence of carcinoma of the prostate is increasing annually by 2-3%. Both race and geographical location have a profound influence of the prevalence of prostate cancer worldwide. Black men in the USA have the highest incidence, while the incidence is much lower in Asian men from China, Japan and Thailand. Although the prostate gland is androgen-dependent, it is now recognized that the biological actions of endocrine-related factors, such as androgens, oestrogens, glucocorticoids and certain dietary and environmental factors, are mediated within the gland by various growth regulatory factors. The growth regulatory factors such as epidermal growth factor (EGF), keratinocyte growth factors (KGF), fibroblast growth factors (FGFs) and insulin-like growth factors II and I are mitogenic and directly stimulate cell proliferation under the modulating influence of steroid hormones. Steroids are therefore essential but not directly responsible for cell proliferation. Certain plant compounds such as isoflavonoids, flavonoids and lignans have been proposed as cancer protective compounds in populations with low incidences of prostate diseases. In particular, soya contains the isoflavone genistein, a compound with many properties which could influence both endocrine and growth factor signalling pathways.

Lignans and isoflavonoids in plasma and prostatic fluid in men: samples from Portugal, Hong Kong, and the United Kingdom.

BACKGROUND: Chinese men have lower incidences of prostate cancer compared to men from Europe and North America. Asians consume large quantities of soya, a rich source of isoflavanoids phyto-oestrogens and have high plasma and urinary levels of these compounds. The mammalian lignans, enterolactone and enterodiol, are another group of weak plant oestrogens and are derived from seeds, cereals and grains. Vegetarians have high plasma and urinary concentrations of lignans. METHODS: The concentrations lignans and isoflavonic phyto-oestrogens were determined by gas chromatography-mass spectrometry (GC-MS) in plasma and prostatic fluid from Portuguese, Chinese and British men consuming their traditional diets. RESULTS: In prostatic fluid the mean concentrations of enterolactone were 31, 162 and 20.3 ng/ml for Hong Kong, Portugal and Britain respectively. Very high levels of enterolactone (> 600 ng/ml) were observed in the prostatic fluid of some of the men from Portugal. High concentrations of equol (3270 ng/ml) and daidzein (532 ng/ml) were found in a sample of prostatic fluid from Hong Kong. Higher mean levels of daidzein were observed in prostatic fluid from Hong Kong at 70 ng/ml, compared to 4.6 and 11.3 ng/ml in samples from Portugal and Britain respectively. Mean levels of daidzein were higher in the plasma samples from Hong Kong (31.3 ng/ml) compared to those from Portugal (1.3 ng/ml) and Britain (8.2 ng/ml). In general, the mean plasma concentrations of enterolactone from the three centres were similar, at 6.2, 3.9 and 3.9 ng/ml in samples from Hong Kong Portugal and Britain respectively. CONCLUSIONS: Higher concentrations of the isoflavanoid phyto-oestrogens, daidzein and equol, were found in the plasma and prostatic fluid of men from Hong Kong compared to those from Britain and Portugal. However, the levels of the lignan, enterolactone, were very much higher in prostatic fluid of Portuguese men. Isoflavanoids and lignans have many interesting properties and may, in part, be responsible for lower incidences of prostate cancer in men from Asia and also some Mediterranean countries. The isoflavanoids from soya, which are present in high concentrations in the prostatic fluid of Asian men, may be protective against prostate disease.

Measurement and metabolism of isoflavonoids and lignans in the human male.

Asian men, who consume a low fat/high fibre soya-based diet, have very much lower incidence of prostate cancer than men from North America and Europe. The soya bean is a rich source of the isoflavonic phyto-oestrogens, daidzein, genistein and equol, compounds which may be cancer-protective in Asian populations. The lignans, enterolactone and enterodiol, plant oestrogens derived from cereals and vegetables, may act in a similar manner in vegetarian men. We report here on the measurement of isoflavonoids and lignans, by gas chromatography-mass spectrometry, in prostatic fluid of men from Asia and Europe and also on the metabolism of these compounds in Western men following dietary supplementation.

Androgens, androgen receptors, antiandrogens and the treatment of prostate cancer.

Androgens play a major role in promoting the growth of the prostate gland. The DHT-androgen receptor complex, by association with the androgen response elements, specifically promotes this androgenic effect on the genome. Also recognized now, however, is that there is a close relationship between this androgen-mediated signalling pathway and those promoted by peptide growth factors, the crosstalk between the pathways being pivotal to growth regulatory control. This is discussed together with the perceived clinical potential of tyrosine kinase inhibitors for the treatment of prostate cancer, when the intracellular signalling, induced by the growth stimulatory factors, can be repressed.

Inhibition of 5 alpha-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids.

Isoflavonoids and lignans, constituents of many plant foods, have been proposed as protective agents in those populations with a low incidence of hormone-dependent cancers. They may act by their inhibition of the metabolism of growth-promoting steroid hormones. This report describes the inhibition of 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase by six isoflavonoids and two lignans in human genital skin fibroblast monolayers and homogenates, and in benign prostatic hyperplasia tissue homogenates. In genital skin fibroblasts, genistein, biochanin A and equol were the most potent inhibitors of 5 alpha-reductase activity, each resulting in greater than 80% inhibition at a concentration of 100 microM. The IC50 values for genistein and a seven-compound mixture were approximately 35 microM and 20 microM (2.9 microM of each compound) respectively. Of the lignans, enterolactone was the most potent inhibitor. Inhibition by biochanin A was shown to be reversible. When genital skin fibroblast homogenates were used, biochanin A was found to inhibit 5 alpha-reductase isozymes 1 and 2 to differing extents (30% and 75% respectively). Genistein was shown to inhibit 5 alpha-reductase 2 in a non-competitive nature (Vmax and Km values without and with inhibitor were 30 and 20 pmol/mg protein per h and 177 and 170 nM respectively). All of the compounds tested inhibited 17 beta-hydroxysteroid dehydrogenase activity in genital skin fibroblast monolayers. When prostate tissue homogenates were used, the compounds tested were better inhibitors of 5 alpha-reductase 1 than 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary flour supplementation decreases post-menopausal hot flushes: effect of soy and wheat.

Plants contain compounds with oestrogen-like action called phytoestrogens. Soy contains daidzin, a potent phytoestrogen, and wheat flour contains less potent enterolactones. We aimed to show in 58 postmenopausal women (age 54, range 30-70 years) with at least 14 hot flushes per week, that their daily diet supplemented with soy flour (n = 28) could reduce flushes compared with wheat flour (n = 30) over 12 weeks when randomised and double blind. Hot flushes significantly decreased in the soy and wheat flour groups (40% and 25% reduction, respectively < 0.001 for both) with a significant rapid response in the soy flour group in 6 weeks (P < 0.001) that continued. Menopausal symptom score decreased significantly in both groups (P < 0.05). Urinary daidzein excretion confirmed compliance. Vaginal cell maturation, plasma lipids and urinary calcium remained unchanged. Serum FSH decreased and urinary hydroxyproline increased in the wheat flour group.