Mariko Uehara

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.

Consumption of wholemeal rye bread increases serum concentrations and urinary excretion of enterolactone compared with consumption of white wheat bread in healthy Finnish men and women.

Rye is an important source of plant lignans in Finland. In the present crossover trial we wanted to study the effect of rye bread as part of the usual diet on serum and urine enterolactone (ENL) concentrations in healthy volunteers. Eighteen men aged 43 (sem 2.0) years and twenty-one women aged 43 (sem 1.6) years consumed wholemeal rye bread and white wheat bread in random order for 4 weeks. The bread periods were separated by a 4 week wash-out period. The breads provided at least 20% of the daily energy intake. The mean intakes of rye bread were 219 (sem 14.6) and 162 (sem 5.3) g/d and those of wheat bread were 200 (sem 9.6) and 153 (sem 5.8) g/d for men and women respectively. Blood samples were collected from all subjects and three 24 h urine samples were collected from ten men and twelve women at the end of both bread periods for the determination of serum concentration and urinary excretion of ENL. The mean serum ENL concentrations in both men and women at the beginning of baseline period and at the end of the rye-bread period remained constant and were significantly higher than those at the end of the wheat-bread period. Correspondingly, daily urinary ENL excretion increased significantly during the rye-bread period compared with the wheat-bread period and was 5- and 10-fold higher in men and women respectively in comparison with the amount of plant lignan precursors measured in the rye bread. These data indicate the presence of other precursors for ENL in rye which are not detected by the current method of measuring plant lignans in food. The possible role of fibre in enhancement of the formation of mammalian lignans from their plant precursors in the gut also remains to be determined.

Time-resolved fluoroimmunoassay of plasma daidzein and genistein

We present a method for the determination of the phytoestrogens daidzein and genistein in plasma (serum). These weakly estrogenic isoflavones occur in soybeans and in smaller amounts in some other beans and plants. It has been suggested that they may afford protection against prostate and breast cancer. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After synthesis of 4-O-carboxymethyl-daidzein and 4-O-carboxymethyl-genistein the compounds are coupled to bovine serum albumin (BSA), then used as antigens to immunize rabbits. The tracers with the europium chelate are synthesized using the same 4-O-derivative of the isoflavones. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). The antisera cross-reacted to some extent with some isoflavonoids but not with flavonoids. The cross-reactivity seems not to influence the results, which were highly specific for both compounds. The correlation coefficients between the TR-FIA methods and the reference method based on isotope dilution gas chromatography-mass spectrometry were high; r-values were about 0.95-0.99 depending on concentration. The intra-assay coefficients of variation (CV%) for daidzein and genistein at three different concentrations vary 3.2-4.5 and 3.2-4.1, respectively. The inter-assay CVs vary 5.0-6.3 and 4.5-5.3, respectively. The working ranges of the daidzein and genistein assays are 1.0-216 and 1.7-370 nmol/l, respectively. The plasma values (n = 80) of daidzein and genistein are very low in Finnish subjects (mean for daidzein, 3.8+/-6.8 and for genistein, 3.2+/-7.6 nmol/l; median value for daidzein 1.5 and for genistein 1.4 nmol/l).

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.

Chronic Phosphorus Supplementation Decreases the Expression of Renal PTH/PTHrP Receptor mRNA in Rats

Dietary intake of high levels of phosphorus is known to increase serum levels of parathyroid hormone (PTH); however, how this increased serum PTH affects the action of PTH in major target tissues, particularly by kidney, remains unknown. In the present study, we therefore undertook to clarify this point in intact animals fed a high-P diet by examining various parameters of PTH action. Twelve weanling Wistar male rats were assigned randomly to two groups: a control group with dietary Ca:P = 1:1 and a high-P group (Ca:P = 1:3) fed the standard AIN-76 diet supplemented with P (0.5 and 1.5 g/100 g of diet). After 3 weeks of feeding, in the high-P diet group, we observed that serum Ca was lowered, without a difference in serum P, when compared to the control group. Excretion of urinary cAMP, an index of renal PTH action, was also decreased, with higher excretion of urinary P in those rats fed the high-P diet. In agreement with the decreased cAMP excretion, a clear reduction in PTH/PTH-related protein (PTHrP) receptor gene expression as estimated by Northern blotting was observed in the kidney, despite increased levels of serum PTH. Thus, the present study indicated that a high-P diet reduces PTH action in the kidney, though the serum PTH is increased.

Change in protein synthesis in rats fed a high phosphorus diet

The concentration of albumin in serum, the level of expression of albumin mRNA, polysome profiles and peptide synthesis activity in the liver were investigated in order to assess the level of protein synthesis in rats fed either a high phosphorus diet containing 1.5% phosphorus or a control diet containing 0.5% phosphorus for 3 weeks. In the rats fed the high phosphorus diet, body weight gain, food efficiency and serum albumin concentration decreased, and the albumin/globulin ratio also decreased compared with the control group. A low level of albumin mRNA expression was observed, consistent with the decrease in serum albumin concentration. The peptide synthesis activity in liver as determined in vitro was lower in the group fed the high phosphorus diet than in the control group. The abundance of heavier polysomes considered to influence protein synthesis in vivo was decreased in the rats given the high phosphorus diet compared with the control group. The results suggest that high phosphorus intake caused a decrease in protein synthesis in liver.