The polymerase chain reaction (PCR) technique was used to investigate the fate of a transgene in the rumen of sheep fed silage and maize grains from an insect-resistant maize line. A 1914-bp DNA fragment containing the entire coding region of the synthetic cryIA(b) gene was still amplifiable from rumen fluid sampled 5 h after feeding maize grains. The same target sequence, however, could not be amplified from rumen fluid sampled from sheep fed silage prepared from the genetically modified maize line. PCR amplification of a shorter (211-bp), yet still highly specific, target sequence was possible with rumen fluid sampled up to 3 and 24 h after feeding silage and maize grains, respectively. These findings indicate that intact transgenes from silage are unlikely to survive significantly in the rumen since a DNA sequence 211-bp long is very unlikely to transmit genetic information. By contrast, DNA in maize grains persists for a significant time and may, therefore, provide a source of transforming DNA in the rumen. In addition, we have examined the biological activity of plasmid DNA that had previously been exposed to the ovine oral cavity. Plasmid extracted from saliva sampled after incubation for 8 min was still capable of transforming competent Escherichia coli to kanamycin resistance, implying that DNA released from the diet within the mouth may retain sufficient biological activity for the transformation of competent oral bacteria.

Responses in variables of amino acid (AA) metabolism across peripheral tissues to feed intake were studied in six sheep (mean live weight 32 kg) prepared with arterio–venous catheters across the hindquarters. Four intakes (0·5, 1·0, 1·5 and 2·5 × maintenance energy) were offered over 2-week periods to each sheep in a Latin square design with two animals replicated. Animals were infused intravenously with a mixture of U-13C-labelled AA for 10 h and integrated blood samples withdrawn from the aorta and vena cava hourly between 5 and 9 h of infusion. Biopsy samples were also taken from skin and m. vastus lateralis. Data from both essential (histidine, isoleucine, leucine, lysine, phenylalanine, threonine) and nonessential (glycine, proline, serine, tyrosine) AA were modelled to give rates of inward and outward transport, protein synthesis and degradation, plus the fraction of total vascular inflow that exchanged with the hindquarter tissues. Rates of inward transport varied more than 10-fold between AA. For all essential AA (plus serine), inward transport increased with food intake (P<0·04). There were corresponding increases in AA efflux (P<0·05) from the tissues for threonine and the branched-chain AA. Protein synthesis rates estimated from the kinetics of these AA also increased with intake (P<0·02). Rates of inward transport greatly exceeded the amount of AA necessary to support protein retention, but were more similar to rates of protein synthesis. Nutritional or other strategies to enhance AA transport into peripheral tissues are unlikely to increase anabolic responses.

Batch cultures of mixed rumen micro-organisms were used to study the effects of different concentrations of malate (Rumalato®; Norel & Nature S.A., Barcelona, Spain; composed of disodium malate–calcium malate (0·16:0·84, w/w)) on the fermentation of four cereal grains (maize, barley, wheat and sorghum). Rumen contents were collected from four Merino sheep fed lucerne hay ad libitum and supplemented with 300 g concentrate/d. Rumalato® was added to the incubation bottles to achieve final concentrations of 0, 4, 7 and 10 mM-MALATE. Gas production was measured at regular intervals up to 120 h. Malate increased (P<0·01) the average fermentation rate of all substrates, and the lag time decreased (P<0·05) linearly with increasing concentrations of malate for all substrates, with the exception of sorghum. in 17 h incubations, the final pH and total volatile fatty acid production increased (P<0·001) linearly for all substrates as malate concentration increased from 0 TO 10 mM. Propionate and butyrate production increased (P<0·05), while the value of the acetate: propionate ratio and L-lactate concentrations decreased (P<0·05) linearly with increasing doses of malate. Malate treatment increased (P<0·05) the CO2 production and decreased the production of CH4, although this effect was not significant (P>0·05) for maize. Malate at 4 and 7 mm increased (P<0·05) optical density of the cultures measured at 600 nm for maize, with no differences for the other substrates. The results indicate that malate may be used as a feed additive for ruminant animals fed high proportions of cereal grains, because it increased pH and propionate production and decreased CH4 production and L-lactate concentrations; however, in general, no beneficial effects of 10 compared with 7 mM-malate were observed.

This present study was designed to investigate the combined modulatory effect of garlic oil (GO) and fish oil (FO) on the antioxidant and drug metabolism systems. Rats were fed either a low-maize oil (MO) diet (50 g MO/kg), high-MO diet (235 g MO/kg) or high-FO diet (205 g FO+30 g MO/kg) and received different doses of GO (0–200 mg/kg body weight) three times per week for 6 weeks. Fatty acid analysis showed that 20: 5n−3 and 22: 6n−3 were incorporated into serum lipid at the expense of 18: 2n−6 and 20: 4n−6 in rats fed the high-FO diet. GO dose-dependently increased hepatic glutathione S-transferase (GST), glutathione reductase, superoxide dismutase (SOD) and ethoxyresorufin O-deethylase (EROD) activities, but decreased glutathione peroxidase and N-nitrosodimethylamine demethylase (NDMAD) activities (P<0·05). With the exception of glutathione peroxidase, the activities of glutathione reductase, SOD, GST, EROD and NDMAD were modulated by the dietary fat. The high-FO group had greater SOD and EROD activity than either MO-fed group; it also had greater NDMAD activity than the low-MO group (P<0·05). GST activity was higher in rats fed high-FO or high-MO diets than rats fed the low-MO diet. Change in erythromycin demethylase activity, however, was not caused by either dietary fat or GO. Immunoblot assay showed that GO dose-dependently enhanced the protein level of the Ya, Yb1, Yc isoenzymes of GST and cytochrome P450 (CYP) 1A1 and 3A1, but GO suppressed CYP2E1 expression. Regardless of the dosage of GO, the high-FO diet increased CYP1A1, CYP3A1 and CYP2E1 levels compared with the high- and low-MO diets. Accompanying the changes observed in immunoblots, CYP1A1 and CYP3A1 mRNA levels were increased by GO in a dose-dependent manner and also increased additively in combination with FO feeding. These present results indicate that co-administration of GO and FO modulates the antioxidant and drug-metabolizing capacity of animals and that the effect of GO and FO on drug-metabolizing enzymes is additive.

Rhubarb (Rheum rhapontiam) stalk fibre was previously shown to be hypolipidaemic under clinical and experimental conditions. The present study was undertaken to investigate whether rhubarb stalk fibre has a hypolipidaemic effect under diabetic conditions. Two models of diabetic rats were used: streptozotocin-induced diabetic rats, and diabetes-prone BB (BBdp) rats. The plasma cholesterol and triacylglycerol concentrations were elevated after the onset of diabetes in BBdp rats, but not in sterptozotocin-induced diabetic rats. The rhubarb-fibre diet had no effect on the plasma cholesterol or triacylglycerol concentrations of diabetic rats. The hypolipidaemic effect of rhubarb stalk fibre has been suggested to be due to the bile-acid-binding capacity of rhubarb fibre, which in turn up regulates cholesterol 7α-hydroxylase (cyp7a) activity. cyp7a is the first and the rate-limiting enzyme in the breakdown of cholesterol to bile acids. We measured the cyp7a activity and mRNA levels in control and diabetic rats fed rhubarb- and cellulose-fibre diets. The cyp7a activity and mRNA abundance were increased in both diabetic rat models, indicating that bile acid synthesis is enhanced in diabetes. Feeding a diet enriched with rhubarb fibre caused a slight but significant increase (P<0·05) in cyp7a enzyme activity in BBdp rats, but no change in cyp7a mRNA abundance was detected. These results suggest that although a rhubarb-fibre-enriched diet increased cyp7a activity in BBdp rats, there was no apparent therapeutic benefit in terms of lowering plasma cholesterol concentrations.

Postprandial concentrations of glucose, insulin and triacylglycerols (TG) correlate to risk for CHD. Carbohydrates affect many metabolites that could have a potential effect on cardiovascular risk factors. The objective of the present study was to examine, using a randomised prospective study, the acute (day 1) and ad libitum medium-term (day 24) effects of four diets: a high-fat diet (HIGH-FAT; 50 % fat, >34 % monounsaturated fatty acids); a low-glycaemic index (GI) diet (LOW-GI; high-carbohydrate, low-GI); a high-sucrose diet (SUCROSE; high carbohydrate increase of 90 g sucrose/d); a high-GI diet (HIGH-GI; high-carbohydrate, high-GI). Daytime profiles (8 h) (breakfast, lunch and tea) of lipid and carbohydrate metabolism were completed during day 1 and day 24. Seventeen middle-aged men with one or more cardiac risk factors completed the study. There was no change from day 1 or between diets in fasting glucose, lipids or homeostatic assessment model (HOMA) on day 24. The HIGH-FAT compared with the three high-carbohydrate diets was associated with lower postprandial insulin and glucose but higher postprandial TG and non-esterified fatty acids (NEFA). There was a significant increase in the 6 h (15.00 hours) TG concentration (day 1, 2·6 (SEM 0·3) MMOL/L v. DAY 24, 3·3 (sem 0·3) mmol/l; P<0·01) on the SUCROSE diet. Postprandial HOMA (i.e. incremental area under the curve (IAUC) glucose (mmol/l per min)×IAUC insulin/22·5 (mU/l per min)) median changes from day 1 to day 24 were −61, −43, −20 and +31 % for the HIGH-FAT, LOW-GI, SUCROSE and HIGH-GI diets respectively. The HIGH-GI percentage change was significantly different from the other three diets (P<0·001). Despite being advised to maintain an identical energy intake there was a significant weight change (−0·27 (sem 0·3) kg; P<0·02) on the LOW-GI diet compared with the SUCROSE diet (+0·84 (sem 0·3) kg). In conclusion the HIGH-FAT diet had a beneficial effect on postprandial glucose and insulin over time but it was associated with higher postprandial concentrations of TG and NEFA. Conversely the HIGH-GI diet appeared to increase postprandial insulin resistance over the study period.

Thirty female Large White × Landrace pigs (average weight 57·2 (SD 1·9) kg) were allocated to one of six dietary treatments containing 0, 1·25, 2·5, 5·0, 7·5 or 10·0 g 55 % conjugated linoleic acids (CLA) isomers (CLA-55)/kg diet and fed for 8 weeks. Each pig was scanned at 0, 28 and 56 d and again at post slaughter using dual-energy X-ray absorptiometry (DXA) to determine the temporal pattern of body composition responses. Values determined by DXA were adjusted using regression equations generated from validation experiments between chemically and DXA-predicted values. Overall, there was a significant linear reduction in fat content with the increasing levels of CLA in the diet (P=0·007, P=0·011, P=0·008 at week 4, week 8 and for the carcass, respectively). The greatest improvement was recorded at the early stages of CLA supplementation and for the highest dose of CLA (week 4, −19·2 % compared with week 8, −13·7 %). In the first 4 weeks of feeding CLA, pigs receiving 10 g CLA-55/kg diet deposited 93 g less fat/d than pigs fed basal diets (P=0·002) compared with only 6 g less fat than control animals in the final 4 weeks. Lean content and lean deposition rate were maximised at 5 and 2·5 g CLA-55/kg diet for the first 4 weeks (P=0·016) and the final 4 weeks of treatment (P=0·17), respectively. DXA estimates of bone mineral content and bone mineral density were not affected by CLA supplementation throughout the experiment. These data demonstrate that dietary CLA decreases body fat in a dose-dependent manner and that the response is greatest over the initial 4 weeks of treatment.

Dietary vitamin A as retinyl ester is hydrolysed and re-esterified with long-chain fatty acids in the small intestine. The esterified vitamin A is subsequently stored in the liver, where it is hydrolysed to free retinol to be transported by carrier proteins to the target tissue. A decreased availability of retinol carrier proteins has been suggested to be responsible for affecting metabolic availability of vitamin A in type 1 diabetes. Using BB Wistar rats, the present study was undertaken to examine whether the presence of a hyperglycaemic state modifies retinyl ester hydrolase (REH) activity in the intestine and the liver. At the onset of diabetes, hepatic REH enzymatic activity was significantly (P<0·05) decreased. However, REH activity remained unaffected in the small intestine, including both ileum and jejunum. Diabetes also resulted in decreased plasma and liver concentrations of retinol. An in vitro study was conducted to examine the effect of diabetes on the intestinal uptake of retinyl palmitate. Jejunum and ileum from diabetic and non-diabetic BB rats were incubated with labelled retinyl palmitate at different concentrations ranging from 32 to 256 nmol/l. The uptake of retinyl palmitate was increased in both diabetic and non-diabetic rats together with the increase of substrate concentration. However, no significant difference was observed in the uptake of retinyl palmitate between diabetic and non-diabetic rats. These present results suggest that the depressed hepatic REH activities may contribute to the diabetes-associated metabolic derangement of vitamin A.

Protein, generally agreed to be the most satiating macronutrient, may differ in its effects on appetite depending on the protein source and variation in digestion and absorption. We investigated the effects of two milk protein types, casein and whey, on food intake and subjective ratings of hunger and fullness, and on postprandial metabolite and gastrointestinal hormone responses. Two studies were undertaken. The first study showed that energy intake from a buffet meal ad libitum was significantly less 90 min after a 1700 kJ liquid preload containing 48 g whey, compared with an equivalent casein preload (P<0·05). In the second study, the same whey preload led to a 28 % increase in postprandial plasma amino acid concentrations over 3 h compared with casein (incremental area under the curve (iAUC), P<0·05). Plasma cholecystokinin (CCK) was increased by 60 % (iAUC, P<0·005), glucagon-like peptide (GLP)-1 by 65 % (iAUC, P<0·05) and glucose-dependent insulinotropic polypeptide by 36 % (iAUC, P<0·01) following the whey preload compared with the casein. Gastric emptying was influenced by protein type as evidenced by differing plasma paracetamol profiles with the two preloads. Greater subjective satiety followed the whey test meal (P<0·05). These results implicate post-absorptive increases in plasma amino acids together with both CCK and GLP-1 as potential mediators of the increased satiety response to whey and emphasise the importance of considering the impact of protein type on the appetite response to a mixed meal.

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.

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.

CHD mortality is extremely low in Japan, particularly in rural districts, when compared with that in Western countries. This has been partly attributed to the difference in dietary lifestyle. We investigated the factors influencing CHD mortality in a rural coastal district of Japan, comprising mercantile, farming, and fishing areas with distinct dietary habits. We prospectively examined the incidence of CHD from 1994 to 1998, as well as coronary risk factors and serum fatty acid concentrations. The incidence of angina pectoris was significantly (P=0·01) lower in the fishing area than in the mercantile and farming areas. Blood pressure, physical activity, prevalence of diabetes, serum levels of uric acid and HDL-cholesterol were similar between the three areas. Total- and LDL-cholesterol levels were significantly lower but the smoking rate was markedly higher in the fishing area than in the other two areas. Serum levels of saturated fatty acids and n−6 polyunsaturated fatty acids (PUFA) were lowest in the fishing area, but n−3 PUFA did not differ significantly. The n−6:n−3 PUFA ratio was lowest and eicosapentaenoic:arachidonic acid was highest in the fishing area. Although many previous studies have emphasized the beneficial effect of n−3 PUFA in preventing CHD, the present study indicated that a lower intake of n−6 PUFA and saturated fatty acids has an additional preventive effect on CHD even when the serum level of n−3 PUFA is high because of high dietary fish consumption.

Functional foods and/or supplements may be used in the context of a healthy lifestyle or as a means to compensate for an unhealthy lifestyle. Adverse long-term and/or cumulative effects of functional food or supplement intake are of public health concern; it is therefore important to identify functional food and supplement users. The present study compared Dutch functional food and supplement consumers with non-consumers with regard to demographic and lifestyle factors. The consumption of the most common functional foods and supplements in 2000 was studied (yoghurt with extra lactic acid bacteria, cholesterol-lowering margarine, lemonade and sweets with extra vitamins and minerals, milk and margarine with extra Ca, Ca tablets, multivitamin and mineral supplements, and Echinacea supplements). Data were obtained from self-administered questionnaires filled in by a consumer panel, aged 19–91 years (response rate 76 %, n 1183), representative of the Dutch population. The number of daily consumers of functional foods or supplements appeared to be relatively low (daily use of multivitamin and mineral supplements, 20 %; all other products, 3–9 %). Explanatory variables depended on the type of product; but gender, age, education, and vegetable intake were significant factors in the logistic regression model. Consumption of cholesterol-lowering margarines was more likely to be reported by individuals with a poorer subjective health (odds ratio 2·62 (95 % CI 1·15, 6·05)) and by smokers (odds ratio 2·93 (95 % CI 1·34, 6·40)). In conclusion, determinants of functional food or supplement use depended on the type of product, so generalisation of consumer characteristics over different foods is not legitimate. In addition to research on lifestyle factors, surveys about consumers' attitudes, norms and knowledge regarding functional foods in relation to actual dietary patterns and health risk profiles are necessary.