These guidelines review the relevant literature on the way plant phyto-oestrogens act on bone and the responsiveness of different bone cell systems to phyto-oestrogenic compounds. The primary emphasis is on the experimental conditions used, the markers available for assessing osteoblast and osteoclast function, and their expected sensitivity. Finally, we assess the published results to derive some general recommendations for in vitro experiments in this area of research.

Little information exists on the extent of day-to-day intra-individual variation in resting metabolic rate (RMR) in women. The present study has investigated the intra-individual variation in RMR of women during the menstrual cycle. Nineteen women (naturally cycling non-pill users) were recruited to the study. Anthropometric and RMR measurements were taken at least three times per week for the duration of one complete menstrual cycle; measurements were taken for a second, consecutive cycle in eight of the nineteen subjects. RMR was measured by indirect calorimetry using a ventilated hood system under standardized conditions. The measurements made throughout each complete menstrual cycle were averaged and the levels of inter- and intra-individual variation in RMR were assessed by determining the CV (%). Mean RMR of the group was 5686 (SD 674) kJ/d; inter-individual variation in RMR was 11·8 %. There were wide differences in the intra-individual variation in RMR of women (CV range 1·7–10·4 %). The CV in ten subjects was small (2–4 %), while the CV in nine women was high (5–10 %), indicating a significant variation in RMR during the menstrual cycle in certain subjects. Using statistical models, it has been shown that there was a significant effect on RMR due to a subject-specific level of variability; this was the case even when accounting for a possible training effect. In conclusion, the findings from our present study show that RMR cannot be assumed to be ‘stable’ in all women. The implications of intra-individual variation in RMR and its impact on energy balance needs further research.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, has been suggested to be a novel risk factor for endothelial dysfunction. It has previously been reported that hyperhomocysteinaemia may be associated with impaired endothelium-dependent vasodilation and reduced plasma level of NO-derived endproducts (NOx). In the present study, plasma levels of arginine and ADMA were measured in twenty-one healthy control subjects, and in twenty-one hyperhomocysteinaemic subjects before and after 6 weeks and 12 months of folic acid supplementation, and compared with previously measured plasma NOx values in the hyperhomocysteinaemic subjects. Compared with control subjects, hyperhomocysteinaemic subjects had higher plasma levels of arginine and ADMA. More importantly, folic acid therapy significantly reduced plasma levels of arginine and ADMA. Furthermore, plasma levels of arginine and ADMA were positively correlated with plasma homocysteine levels and negatively correlated with plasma folate, as well as negatively correlated with plasma NOx. Our results suggest that ADMA may be a mediator of the atherogenic effects of homocysteine.

Subjects with insulin resistance have been shown to have higher storage levels of intramyocellular lipid (IMCL) than their insulin-sensitive counterparts. It has been proposed that elevated IMCL stores may be the main cause of insulin resistance. The aim of the present study was to ascertain whether there is a causal relationship between IMCL storage and insulin resistance. IMCL storage was assessed using magnetic resonance spectroscopy and insulin sensitivity was assessed by performing an oral glucose tolerance test. A 4-week intervention of reduction of dietary glycaemic index was used to manipulate insulin sensitivity in a cohort of healthy volunteers; the effects of this intervention on IMCL were measured after 4 weeks of intervention. Significant improvements in the insulin sensitivity index occurred following the dietary intervention (baseline 7·8 (sem 1·11) v. post-intervention 9·7 (sem 1·11), P=0·02). However, there were no changes in IMCL storage levels, suggesting that insulin sensitivity can be manipulated independently of IMCL. This suggests that in healthy volunteers, insulin sensitivity is independent of IMCL storage and the high storage levels that have been found in insulin-resistant subjects may occur as a consequence rather than a cause of insulin resistance.

In the present study we examined whether two virgin olive oils (VOO1 and VOO2), of the same variety (Olea europaea var. hojiblanca) and with a similar composition of minor components but differing in the content of triacylglycerol molecular species, had different effects on blood pressure and plasma lipid levels in a healthy elderly population. Thirty-one participants, aged 84·9 (SD 6·4) years, were asked to participate in the study. No differences were found with regard to blood pressure after both experimental periods (VOO1 and VOO2). However, plasma total cholesterol and LDL-cholesterol were reduced only after VOO1 (P<0·01). The reduction of plasma cholesterol concentrations was related to the incorporation of oleic acid into plasma cholesteryl esters and phospholipids, which was higher after VOO1 (P<0·01). Indeed, the oleic acid concentration in cholesteryl esters and phospholipids strongly correlated with plasma total cholesterol and LDL-cholesterol levels in all experimental periods studied (r2>0·418, P<0·07), except for phospholipids in VOO1 (P=0·130 for total cholesterol and P=0·360 for LDL-cholesterol). These results have demonstrated that blood pressure and plasma lipids can be modified by the consumption of VOO in elderly people, but that the extent of such modification depends on the composition and amount of active minor components and triacylglycerol molecular species.

In the context of the Greek segment of the European Prospective Investigation into Cancer and Nutrition (EPIC) we have conducted a cross-sectional study to determine the effect of demographic, lifestyle and nutritional factors of plant origin on retinol, α-tocopherol and γ-tocopherol. Study subjects were a random sample of forty-five men and sixty-eight women, aged 30–82 years, from the Greek EPIC cohort of 27953 volunteers. Analyses were done using linear regression models with the vitamin blood levels as dependent variables, and BMI, the intake of selected food items, total energy intake and a set of demographic factors as independent variables. Women had significantly lower plasma retinol levels (P<0·006) than men. Both α- and γ-tocopherol levels increased by approximately 5 % for every additional 5 years of age. Among dietary factors, intake of non-fruity vegetables was associated with increase, while intake of added lipids other than olive oil with decrease, of plasma retinol levels. BMI was positively associated with plasma γ-tocopherol only, while none of the foods or food groups investigated significantly affected the plasma levels of either of the two tocopherols. These findings need to be confirmed before attempts are made to use them in the context of explanatory mechanistic processes.

The aim of the present study was to determine the effects of age and diet (breast milk, formula milk and weaning diet) on metabolic activities in faecal samples from infants aged 1 week to 1 year, and to compare these findings with activities found in samples from adults. Such activities can provide valuable information on functional changes in the microbiota that may have significance for the health of the host. Fresh faecal samples were collected from forty-four breast-fed infants (twenty-four males, twenty females) and thirteen formula-fed infants (three males, ten females) throughout the first year of life. The samples were analysed for protein-breakdown products, including the faecal concentrations of NH3, phenol and p-cresol, and faecal bacterial enzyme activities. There was wide individual variation in all variables measured; however, the values in infants were substantially lower then those found in adults. In pre-weaned infants, faecal NH3 concentration and β-glucuronidase activity were the only endpoints that were significantly different in breast-fed and formula-fed infants (P<0·001 and P<0·05 respectively). This was not apparent after weaning. There was a significant difference between the breast-fed and formula-fed weaned groups and their pre-weaned counterparts only for NH3 (P<0·05). β-Glucuronidase activity and phenol concentration were significantly (P<0·01) greater in weaned breast-fed infants compared with pre-weaned breast-fed infants. No differences were observed between pre-weaned and weaned formula-fed infants for any of the variables except for NH3 concentration. It can be concluded from the present study that there are significant differences in two faecal characteristics between breast- and formula-fed infants and that changes occur as the infants grow older and are weaned onto solid foods.

Osteoporosis has become a major public health problem. Because the biggest culprit in the process of bone loss is oestrogen deficiency, hormone replacement therapy remains the mainstay for prevention, but prophylaxis by this means is limited. Phyto-oestrogens deserve special mention because emerging data support the suggestion that these weakly oestrogenic compounds, present in plants, may prevent bone loss associated with the menopause and thus represent a potential alternative therapy for a range of hormone-dependent conditions, including postmenopausal symptoms. A substantial body of work in animal models in the past few years has provided convincing data for significant improvements in bone mass and other endpoints following feeding with soya. Thus, phyto-oestrogens appear to have potential promise for maintaining or modestly improving bone mass of human subjects when consumed at optimal dosages. However, we must appreciate the limits of the information reached before extrapolating to man and we need to gather more data before health professionals can actively advocate the increased consumption of soya. Indeed, it will be important further to characterise the physiological effects of phytooestrogens and their margins of safety.

Capric (10:0) and lauric (12:0) acid and conjugated linoleic acid (CLA) have been shown to increase paracellular permeability across human intestinal-like Caco-2 cell monolayers. While this has generated interest in terms of improved drug absorption and delivery, little has been done in terms of their potential effect on nutrient transport across the intestinal epithelium. Therefore, the objective of the present study was to investigate the effect of these fatty acids on transepithelial Ca transport in Caco-2 cells. Cells were seeded onto permeable transport membranes and allowed to differentiate, over 21 d, into intestinal-like cell monolayers. Monolayers (n 9 per treatment) were exposed to 0 (control) or 80 μM-10:0, 80 μM-12:0, 80 μM-18:2, 80 μM-CLA (mixed isomers), 80 μM-cis-9,trans-11 CLA or 80 μM-trans-10,cis-12 CLA for 22 d after seeding (chronic effect) or for 24 h before Ca transport studies (acute effect) on day 22. After exposure, transepithelial and transcellular transport of 45Ca, fluorescein transport (a marker of paracellular Ca transport) and transepithelial electrical resistance (TEER, an indicator of permeability) were measured. Overall Ca transport and TEER in Caco-2 cells were unaffected by exposure to any of the fatty acids for 24 h, or to 18:2, CLA blend or cis-9,trans-11 CLA for 22 d. Paracellular (but not total transepithelial and transcellular) Ca transport across Caco-2 cells was significantly increased (P<0·01, by about 1·5-fold relative to the control value) by exposure to 10:0, 12:0 and trans-10,cis-12 CLA for 22 d, suggesting that these non-esterified fatty acids could potentially enhance Ca absorption in vivo.

Ornithine α-ketoglutarate (OKG) administration in human subjects elicits insulin secretion. We investigated whether this action was related to an effect of OKG on islets of Langerhans, and addressed the underlying mechanisms of action. For this purpose the influence of OKG on insulin secretion was measured in isolated rat islets of Langerhans under two different conditions. In incubated islets, OKG (0·25 to 2·5 mmol/l) significantly and dose-relatedly increased insulin secretion (1·7- to 4·2-fold; P<0·05 v. basal). To study the kinetics of OKG-stimulated insulin secretion, perifusion experiments were performed, which showed that OKG affected insulin secretion in both initial and later phases. Experiments using α-ketoglutarate (α-KG) (1 mmol/l) or ornithine (Orn) (2 mmol/l) alone, in concentrations equal to that of OKG, showed that the OKG-induced insulin secretion could not be obtained by either component alone, suggesting that an α-KG–Orn interaction is mandatory for the insulin-secreting effect to occur. Since data obtained in vivo suggest that effects of OKG may depend on the synthesis of NO, glutamine and/or polyamines, three metabolic pathways potentially involved in insulin secretion, we then evaluated their contribution by means of their respective inhibitors: α-NG-nitroarginine methyl ester (l-NAME), methionine sulfoximine (MSO) and difluoromethylornithine (DFMO). Both l-NAME and MSO were able significantly to reduce OKG-induced insulin secretion (30 and 40 % respectively; P<0·05), while DFMO was ineffective. Thus OKG is an effective stimulator of insulin secretion, requiring the joint presence of both Orn and α-KG, and acting mainly via the synthesis of NO and glutamine. A better understanding of OKG insulino-secretory properties and its mechanisms of action are a prerequisite for its use in insulin-compromised situations.

Chronic energy restriction, α-tocopherol supplementation and their interaction with exhaustive exercise were investigated. Eleven-week-old male Wistar rats (n 6×10) were fed either a control (C), a 30 % carbohydrate-energy-restricted control (R) or an α-tocopherol-supplemented (S) diet for 5 months. The animals in each diet were divided into exercised (E) and non-exercised (NE) groups. Before killing, the exercised rats were required to run to exhaustion (39 (SE 6), 69 (se 11) and 18 (se 2) min for the C, R and S groups, respectively). Lipid peroxidation (thiobarbituric acid-reactive substances; TBARS), protein damage (reactive carbonyls) and α-tocopherol were determined in gastrocnemius, liver, brain an/r plasma. There was no difference in lipid peroxidation between the R and C groups, but in liver and muscle peroxidation appeared significantly lower in the S than the other two diets. TBARS in the brain were similar in all groups. On the other hand, reactive carbonyls showed that both the R and S diets reduced protein damage in the brain, while exhaustive exercise increased it. For liver and muscle, however, reactive carbonyl levels were similar in all groups. α-Tocopherol supplementation increased the vitamin concentrations in liver, muscle and plasma, but exercise decreased them in plasma and brain. Carbohydrate-energy restriction increased (P=0·0025) resistance to exhaustive exercise considerably without depleting stores of α-tocopherol or exacerbating oxidative damage in monitored tissues. It is concluded that while exhaustive exercise promotes a tissue-specific oxidative damage detectable only in brain proteins, both experimental diets tended to ameliorate this condition.

Research on the bone effects of natural phyto-oestrogens after menopause is at a relatively early stage. Published studies are few, difficult to compare and often inconclusive, due in part to design weaknesses. Currently, many questions remain to be answered including to what extent a safe daily intake may prevent postmenopausal bone loss. These questions can only be addressed by conducting well-planned, randomised clinical trials that take into consideration present knowledge in the oestrogen, phyto-oestrogen and bone fields. This review is intended to provide hints for critical decision-making about the selection of subjects, type of intervention, suitable outcome measures and variables that need to be controlled.

C-reactive protein (CRP) is a sensitive marker for low-grade inflammation. Long-chain n-3 polyunsaturated fatty acids (PUFA) have anti-inflammatory effects. The objective of the present study was to investigate the effect on serum levels of CRP of n-3 PUFA at two different doses. We also investigated correlations between CRP and the cellular contents of PUFA. Sixty healthy volunteers (twenty-five women and thirty-five men) were randomly assigned to three treatment groups in a double-blind design. The subjects received a supplement of either 6·6 g n-3 PUFA/d, 2·0 g n-3 PUFA/d or placebo (olive oil) for 12 weeks. CRP was measured using a highly sensitive assay. The median serum CRP concentration was 0·78 mg/l. No significant correlations were found between CRP and the content of n-3 PUFA in granulocytes or platelets. Subjects receiving n-3 PUFA had a significant (P<0·01) increase in the cellular contents of 20: 5n-3, 22: 5n-3 and 22: 6n-3, with the largest increase occurring in the group receiving 6·6 g PUFA/d. A significant (P<0·01) decrease in cellular content of 18: 2n-6 and 20: 4n-6 was observed simultaneously. Serum CRP concentrations, however, were unaffected by the PUFA-containing supplements. The present study shows that dietary supplementation with PUFA-containing supplements has no effect on serum concentrations of CRP, measured with a highly sensitive assay, in healthy subjects.

To explore the hypothesis that proliferating lymphoid cells in immune-stimulated lymph nodes obtain nutrients locally from adjacent adipose tissue, adult guinea pigs were fed for 6 weeks on standard chow or on chow supplemented with 100 g suet, sunflower oil or fish oi/g. All the guinea pigs ate standard chow for the last 5 d, during which swelling of one popliteal lymph node was stimulated by repeated local injection of lipopolysaccharide. The fatty acid compositions of phospholipids in both popliteal and in several mesenteric lymph nodes, and of triacylglycerols in eleven samples of adipose tissue defined by their anatomical relations to lymph nodes, were determined by GC. The proportions of fatty acids in the phospholipids extracted from the stimulated popliteal node correlated best with those of triacylglycerols in the surrounding adipocytes, less strongly with those of adipocytes elsewhere in depots associated with lymphoid tissue, but not with those of nodeless depots. The composition of triacylglycerols in the perinodal adipose tissue changed under local immune stimulation. We conclude that proliferating lymphoid cells in activated lymph nodes obtain fatty acids mainly from the triacylglycerols in adjacent perinodal adipose tissue. Immune stimulation prompts changes in the fatty acid composition of the triacylglycerols of adipocytes in node-containing depots that equip the adipose tissue for provisioning immune responses. Such local interactions show that specialised adipocytes can act as an interface between whole-body and cellular nutrition, and may explain why mammalian adipose tissue is partitioned into a few large and many small depots.

The effect of vitamin E derivatives on the urokinase-plasminogen activator (u-PA) system of resting and phorbol myristate acetate (PMA)-activated ovine macrophages and neutrophils were investigated. Blood monocyte–macrophages and neutrophils were isolated from twenty-four animals. Macrophages or neutrophils were cultured in vitro for 3 or 24 h with or without various vitamin E derivatives: free α-tocopherol (α-T), α-tocopheryl acetate (α-TA), or α-tocopheryl succinate (α-TS). Following incubation, cells were stimulated with 80 μM-PMA. Total cell-associated u-PA, membrane-bound u-PA and free u-PA binding sites were determined before and after stimulation with PMA. Results showed that none of the vitamin E derivatives had any effect (P>0·05) on the u-PA system of resting monocyte–macrophages or neutrophils. In contrast, α-TS, but not α-TA or α-T, increased (P<0·01) total cell-associated u-PA and membrane-bound u-PA of PMA-stimulated macrophages and neutrophils. α-TS had no effect (P>0·05) on total u-PA and membrane-bound u-PA activities of macrophages and neutrophils cultured in the presence of 4-phorbol 12,13 didecanoate, a phorbol ester that does not activate protein kinase (PK) C. Addition of H7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), which is a potent inhibitor of both PK A and C, completely abolished the effect of α-TS on total cell-associated u-PA and membrane-bound u-PA of PMA-activated macrophages and neutrophils. Addition of HA1004 (N-(2-quanidinoethyl)-5-isoquinoline sulfonamide hydrochloride), which is a potent PK A but a weak PK C inhibitor, had no effect (P>0·05) on total cell-associated u-PA and membrane-bound u-PA of PMA-activated macrophages and neutrophils cultured in the presence of α-TS. Thus, PK C modulates the effect of α-TS on the u-PA system of ovine macrophages and neutrophils.

Although food supply statistics are commonly used in ecological studies of diet and disease, little information is available on how they compare with reported intakes of foods. The objective of the present study was to compare fruit and vegetable availability with estimates of national mean intakes derived from national food consumption surveys. Food availability statistics from the FAO were used. For each country, mean national supply, based on at least 3 years of FAO data, was calculated. National estimates of mean fruit and vegetable intakes were derived from population-based surveys from fifteen countries, gathered for the World Health Organization Global Burden of Disease Study revision for 2000. Extrapolations were made when survey data did not cover all age groups. For each country, the FAO:survey estimate ratio was calculated. This ratio ranged from 0·93 to 2·70 (median value=1·39). Although there was a tendency for FAO data to overestimate intakes (fourteen out of fifteen countries), the degree of overestimation varied greatly among the countries included in this study (5–270 %). As food supply statistics are the only source of information on dietary patterns in most countries of the world, further information on how they reflect food intakes is needed. Obtaining detailed and valid estimates of dietary intakes in more countries around the world will be essential for such comparisons.

Digestion and portal net flux of nutrients were studied in sheep fed twice daily with fresh orchard-grass. Digestive flows were measured in six fistulated sheep using the double-marker technique. Three sheep were fitted with catheters and blood-flow probes, allowing nutrient net flux measurements across the portal-drained viscera (PDV), the mesenteric-drained viscera (MDV) and the rumen. Total tract apparent digestion of N was similar to portal net appearance of N, calculated as the sum of free amino acids (FAA), peptide amino acids (PAA), NH3, and urea net fluxes. PAA accounted for 25 % of non-protein amino acid net release across the PDV. With the exception of glycine and glutamate, the small intestine was the main contributor to this PAA net release. The essential amino acid (EAA) apparent disappearance between the duodenum and the ileum was lower than the net appearance of EAA (FAA + PAA) across the MDV. The value of PDV:MDV flux of free EAA was, on average, 78 %. The rumen accounted for 30 % of the net uptake of EAA by the PDV tissues not drained by the mesenteric vein. Rumen net release of acetate, propionate, butyrate, 3-hydroxybutyrate, and lactate accounted for 70, 55, 46, 77 and 52 %, respectively, of their portal net releases. Conversely, the small intestine was a net consumer of arterial acetate and 3-hydroxybutyrate. Dynamic study of nutrient net fluxes across the PDV showed that throughout a feeding cycle, the liver faced a constant flux of amino acids (AA), whereas volatile fatty acid and NH3 net fluxes varied in response to the meal. The present study specified, in forage-fed sheep, the partitioning of nutrient net fluxes across the PDV and the role of peptides in portal net release of AA.

Hormone replacement therapy (HRT) for preventing loss of bone following the menopause is utilised by only 8–10% of possible users, largely due to a fear of increased risk of breast cancer. Plant oestrogen-like compounds (phyto-oestrogens) have been proposed as an alternative to HRT to prevent osteoporosis. One class of phyto-oestrogens (the isoflavones) is found in soya foods and red clover. The food industry is developing a wide variety of new foods containing soya to substantially increase isoflavone intake, as well as extracting isoflavones from soya and clover to use as additives to non-soya foods. Pharmaceutical companies are also preparing isoflavone extracts to be used in pill form. In each case the targeted delivery is ˜50mg of isoflavones/d. Is this dose of isoflavones safe? In this review of the current literature, it is concluded that isoflavones consumed orally and in doses below 2mg/kg body weight per d should be considered safe for most population groups. Whether these doses are sufficient to prevent osteoporosis is a separate matter.

The UK Food Standards Agency (FSA) convened a group of expert scientists to discuss and review UK FSA- and Department of Health-funded research on diet and bone health. This research focused on the lifestyle factors that are amenable to change and may significantly affect bone health and the risk of osteoporotic fracture. The potential benefits of fruits and vegetables, meat, Ca, vitamins D and K and phyto-oestrogens were presented and discussed. Other lifestyle factors were also discussed, particularly the effect of physical activity and possible gene–nutrient interactions affecting bone health.

Considerable evidence exists that smoking significantly lowers the concentration of plasma antioxidants. While for most antioxidants this effect appears to result mainly from altered dietary habits, ascorbic acid has recently been shown to be depleted by smoking per se. However, the direct cause of ascorbate depletion remains unclear. Erythorbic acid is a stereoisomer of ascorbic acid commonly used as antioxidant in foodstuffs and has the same redox properties as ascorbic acid. We therefore investigated if erythorbic acid could be used as a non-isotopic marker of smoking-induced oxidative stress. In a sample of smokers (n 10) and non-smokers (n 10), the pharmacokinetics of erythorbic acid were followed after a single oral dose (1 g) and subsequently, the effect of a 2-week ascorbic acid supplementation (0·5 g/d) on erythorbic acid kinetics was studied in a double-blind, placebo-controlled fashion. There were no significant effects of smoking or supplementation on relative bioavailability (difference in area under curve, AUC0-∞) of erythorbic acid (smokers 357 (SD 119), non-smokers 414 (SD 142) μmol·h/l; P=0·34). Time to reach maximum plasma concentration (Tmax) was significantly less in smokers (P=0·03). If the relative pharmacokinetics of erythorbic acid between smokers and non-smokers compares with those of AA, our present results do not suggest that altered pharmacokinetics is likely to play a major role in the ascorbic acid depletion consistently observed in smokers.