Microarrays represent a powerful tool for studies of diet–gene interactions. Their use is, however, associated with a number of technical challenges and potential pitfalls. The cost of microarrays continues to drop but is still comparatively high. This, coupled with the complex logistical issues associated with performing nutritional microarray studies, often means that compromises have to be made in the number and type of samples analysed. Additionally, technical variations between array platforms and analytical procedures will almost inevitably lead to differences in the transcriptional responses observed. Consequently, conflicting data may be produced, important effects may be missed and/or false leads generated (e.g. apparent patterns of differential gene regulation that ultimately prove to be incorrect or not significant). This is likely to be particularly true in the field of nutrition, in which we expect that many dietary bioactive agents at nutritionally relevant concentrations will elicit subtle changes in gene transcription that may be critically important in biological terms but will be difficult to detect reliably. Thus, great care should always be taken in designing and executing microarray studies. This article seeks to provide an overview of both the main practical and theoretical considerations in microarray use that represent potential sources of technical variation and error. Wherever possible, recommendations are made on what we propose to be the best approach. The overall aims are to provide a basic framework of advice for researchers who are new to the use of microarrays and to promote a discussion of standardisation and best practice in the field.

Conjugated linolenic acid (CLN) refers to a group of octadecatrienoic acid isomers that have three double bonds in conjugation. Both pomegranate and tung seed oils are rich in CLN but the major isomer in the former is cis9,trans11,cis13 while in the latter it is cis9,trans11,trans13. The present study examined the effects of CLN, isolated from either pomegranate seed oil or tung seed oil, and α-linolenic acid (LN), isolated from flaxseed oil, on serum cholesterol levels in male hamsters (body weight 105 g; age 10 weeks) fed a 0.1 % cholesterol and 10 % lard diet, for a period of 6 weeks. All hamsters were allowed free access to food and fluid. The blood samples were taken by bleeding from the retro-orbital sinus into a heparinized capillary tube under light ether anaesthesia after overnight fasting at weeks 0, 2, 4 and 6. It was found that supplementation of CLN at levels of 12.2–12.7 g/kg diet exhibited no significant effect on serum cholesterol level while LN at a similar level of supplementation had serum cholesterol reduced by 17–21 % compared with the control diet containing no LN and CLN. Supplementation of CLN and LN significantly decreased hepatic cholesterol but no effect was observed on heart and kidney cholesterol levels. It was concluded that LN possessed hypocholesterolaemic activity while CLN had no effect on blood cholesterol, at least in hamsters.

We investigated the effect of different types of dietary protein on glycogen content in liver and skeletal muscle of exercise-trained rats. Twenty-four male Sprague-Dawley rats (approximately 100 g; n 6 per group) were divided into sedentary or exercise-trained groups with each group being fed either casein or whey protein as the source of dietary protein. Rats in the exercised groups were trained during 2 weeks using swimming exercise for 120 min/d, 6 d/week. Exercise training resulted in an increase in the skeletal muscle glycogen content. Furthermore, the whey protein group significantly increased the skeletal muscle glycogen content compared with the casein group. The increase in glycogen content in liver was significantly greater in rats fed the whey protein diet compared with those fed the casein diet. We also found that the whey protein diet increased the activity of liver glucokinase, whereas it decreased the activities of 6-phosphofructokinase and pyruvate kinase compared with the casein diet. However, hepatic total glycogen synthase activity and mRNA expression were similar with the two diets. In the skeletal muscle, whey protein decreased only 6-phosphofructokinase activity compared with casein. Total glycogen synthase activity in the skeletal muscle in the whey protein group was significantly higher than that in the casein group. The present study is the first to demonstrate that a diet based on whey protein may increase glycogen content in liver and skeletal muscle of exercise-trained rats. We also observed that whey protein regulated glycogen metabolism in these two tissues by different mechanisms.

In pigs the extensive threonine utilization by the splanchnic tissues explains the relative inefficiency of dietary threonine conversion for body protein accretion. Two experiments were conducted to estimate the contribution of the portal drained viscera (PDV) and the liver to threonine metabolism and especially catabolism in growing pigs. In the first experiment, four pigs were surgically prepared for chronic catheter insertion in the portal, hepatic and jugular veins and in the carotid artery. They were continuously infused with l-[1-13C]threonine through the jugular catheter. The PDV and total splanchnic viscera (PDV and the liver) extracted 14·3 and 18·8 % of arterial threonine input, respectively. In a second experiment, we studied the metabolism of dietary threonine in the PDV and the liver in six female growing pigs. Animals were surgically prepared as in the first experiment except that l-[1-13C]threonine and [15N]glycine were continuously infused in the duodenum for 10 h. Unlabelled and labelled threonine and glycine PDV, liver and splanchnic tissues balance were calculated from plasma samples taken during the last 2 h of this infusion. Splanchnic tissues extracted 60 % of infused labelled threonine, 88 % of which was extracted by PDV so that threonine extraction by the liver was low. Both the liver and the pancreas can degrade threonine through the l-threonine 3-dehydrogenase pathway but not the intestine. Our data suggest that threonine catabolism through the l-threonine 3-dehydrogenase pathway was only a minor component of total threonine utilization in the splanchnic tissues.

We investigated the feasibility of increasing ursodeoxycholic acid (UDCA) in the enterohepatic circulation of pigs by administering living bacteria capable of epimerising endogenous amidated chenodeoxycholic acid (CDCA) to UDCA. We first demonstrated that combining Bifidobacterium animalis DN-173 010, as a bile salt-hydrolysing bacterium, and Clostridium absonum ATCC 27555, as a CDCA to UDCA epimerising bacterium, led to the efficient epimerisation of glyco- and tauro-CDCA in vitro, with respective UDCA yields of 55·8 (se 2·8) and 36·6 (se 1·5)%. This strain combination was then administered to hypercholesterolaemic pigs over a 3-week period, as two daily preprandial doses of either viable (six experimental pigs) or heat-inactivated bacteria (six controls). The main effects of treatment were on unconjugated bile acids (P=0·035) and UDCA (P<0·0001) absorbed into the portal vein, which increased 1·6–1·7- and 3·5–7·5-fold, respectively, under administration of living compared with inactivated bacteria. In bile, UDCA did not increase significantly, but the increase in biliary lithocholic acid with time in the controls was not observed in the experimental pigs (P=0·007), and the same trend was observed in faeces. All other variables (biliary lipid equilibrium, plasma lipid levels and partition of cholesterol between the different lipoprotein classes) remained unaffected by treatment throughout the duration of the experiment. In conclusion, it is feasible to increase the bioavailability of UDCA to the intestine and the liver by administering active bacteria. This may represent an interesting new probiotic activity, provided that in future it could be expressed by a safe food micro-organism.

An adverse environmental experience of the growing fetus may lead to permanent changes in the structure and function of organs that may predispose the individual to chronic diseases in later life; however, nothing is known about the occurrence and mechanisms of heart failure. We employed a rat model in which pregnant dams were fed diets containing either 180 g (normal) or 90 g (low) casein/kg for 2 weeks before mating and throughout pregnancy. The ejection fraction (EF) of the pups exposed to the low-protein (LP) diet was severely depressed in the first 2 weeks of life and was associated with an increase in cardiomyocyte apoptosis. This early depressed cardiac function was followed by progressive recovery and normalization of the EF of the offspring in the LP group. The left ventricular (LV) internal diameters were increased between 24 h and 84 d (12 weeks) of age in the LP-exposed group. Although between 3 d and 2 weeks of age the LV wall of the heart in the LP group was thinner, a progressive increase in LV wall thickness was seen. At 40 weeks of age, although the EF was normal, a two-fold elevation in LV end-diastolic pressure, reduced cardiac output, decreased maximum rates of contraction and relaxation, and reduced mean arterial pressure were observed. Our findings demonstrate that exposure of the developing fetus to a maternal LP diet programs cardiac dysfunction in the offspring in later life.

Two incubation trials were carried out with the rumen-simulation technique (RUSITEC). In each trial, four vessels received a diet of grass hay and concentrate (600 and 400 g/kg DM, respectively; diet F), and the other four were fed a diet composed of concentrate and barley straw (900 and 100 g/kg DM, respectively; Diet C). Vessels were given 20 g of the corresponding diet daily, and half of them were supplemented with disodium malate to achieve a final concentration of 6.55 mM. There were no effects (P>0·05) of malate either on pH or on the daily production of NH3-N, but malate treatment increased (P<0·05) DM, neutral detergent and acid detergent fibre disappearance after 48 h incubation. The daily production of propionate and butyrate increased (P<0·001), and the ratio CH4:volatile fatty acids decreased (P<0·001) by supplementing both diets with malate. Whereas adding malate to the F diet produced an increase in acetate production (P=0·011) and the growth of solid-associated micro-organisms (P=0·037), no effects (P>0·05) were observed for diet C. For both diets, there were no differences (P>0·05) between treatments in the daily flow of liquid-associated micro-organisms measured using 15N as a microbial marker. These results indicate that malate stimulated the in vitro fermentation of both diets by increasing the apparent disappearance of the diet and decreasing the ratio of CH4:volatile fatty acids, but a greater response was observed with diet F. If these results are confirmed in vivo, malate could be used as a feed additive for ruminants fed diets containing medium proportions of forage (i.e. dairy animals) and not only in animals fed high-concentrate diets, as has so far been proposed.

A recent study from our laboratory has shown that a mixture of glucose and fructose ingested at a rate of 1·8 g/min leads to peak oxidation rates of approximately 1·3 g/min and results in approximately 55 % higher exogenous carbohydrate (CHO) oxidation rates compared with the ingestion of an isocaloric amount of glucose. The aim of the present study was to investigate whether a mixture of glucose and fructose when ingested at a high rate (2·4 g/min) would lead to even higher exogenous CHO oxidation rates (>1·3 g/min).Eight trained male cyclists (VO2max: 68±1 ml/kg per min) cycled on three different occasions for 150 min at 50 % of maximal power output (60±1 % VO2max) and consumed either water (WAT) or a CHO solution providing 1·2 g/min glucose (GLU) or 1.2 g/min glucose+1·2 g/min fructose (GLU+FRUC). Peak exogenous CHO oxidation rates were higher (P<0·01) in the GLU+FRUC trial compared with the GLU trial (1·75 (se 0·11) and 1·06 (se 0·05) g/min, respectively). Furthermore, exogenous CHO oxidation rates during the last 90 min of exercise were approximately 50 % higher (P<0·05) in GLU+FRUC compared with GLU (1·49 (se 0·08) and 0·99 (se 0·06) g/min, respectively). The results demonstrate that when a mixture of glucose and fructose is ingested at high rates (2·4 g/min) during 150 min of cycling exercise, exogenous CHO oxidation rates reach peak values of approximately 1·75 g/min.

Lactating mammals usually exhibit a breakdown of immunity to parasites, i.e. they have larger worm burdens than their non-lactating counterparts. Here, we tested the hypothesis that a secondary infection with Nippostrongylus brasiliensis in lactating rats is sensitive to dietary protein content. We also tested whether this infection affects host food intake. Rats either remained uninfected throughout the study or were given a single infection before mating (primary infection) and re-infected on day 2 of lactation (secondary infection) with 1600 infective larvae. Infected rats were fed foods during lactation formulated to supply 100 (low protein; LP), 200 (medium protein; MP) or 300 (high protein; HP) g crude protein per kg DM; non-infected rats were fed either the LP or HP food. Litter size was standardized to ten pups between parturition (day 0) and secondary infection (day 2). Ten days after secondary infection, MP and HP rats had excreted fewer nematode eggs, and had fewer adult nematodes in their small intestine and nematode eggs in their colon than the LP rats. Primary infection increased food intake in late pregnancy, and increased maternal body weight and litter size at parturition. Secondary infection did not affect mean food intake, maternal and litter weight, although food intake was reduced for 1 d following infection. These results support the view that a secondary infection with N. brasiliensis is sensitive to dietary protein content, and that the latter infection does not impair lactational performance. Future studies may focus on elucidating the nutritional sensitivity of immune responses underlying the reduced secondary N. brasiliensis infection.

Vitamin C (ascorbate) is essential for hydroxylation of prolyl and lysyl residues in nascent collagen, the failure of which leads to connective tissue lesions of scurvy. Of the pyridinium-type cross-links in mature collagen, pyridinoline requires more hydroxylysyl residues than does deoxypyridinoline. Our study tested the hypothesis that pyridinoline:deoxypyridinoline ratios in urinary degradation products may vary with ascorbate status in man. These ratios were compared between British and Gambian prepubertal boys, mean age 8·3 years, and in Gambian boys between two seasons with contrasting ascorbate availability. The mean cross-links ratio in 216 British boys was 4·36 (sd 0·71), significantly greater (P<0·0001) than in sixty-two Gambian boys: 3·83 (sd 0·52). In the Gambians the cross-links ratio was significantly higher in the dry season (with high ascorbate intake and status) than in the rains (with low intake and status). A 7-week controlled intervention was carried out in Gambian boys during the rainy season (the ‘hungry’ season, when vitamin C-containing foods are virtually unavailable): 100 mg ascorbate/d was given to one group of thirty-two Gambian boys and placebo to another group. The intervention did not, however, significantly alter the cross-link ratio, possibly because the response time and/or intervention–response delay is >7 weeks. If confirmed, the putative association between ascorbate and collagen cross-link ratios in man could become the basis for a functional test for adequacy of ascorbate status.

After resective and reconstructive surgery in the gastrointestinal tract, oral feeding is traditionally avoided in order to minimize strain to the anastomoses and to reduce the inherent risks of the postoperatively impaired gastrointestinal motility. However, studies have given evidence that the small bowel recovers its ability to absorb nutrients almost immediately following surgery, even in the absence of peristalsis, and that early enteral feeding would preserve both the integrity of gut mucosa and its immunological function. The aim of this study was to investigate the impact of early enteral feeding on the postoperative course following oesophagectomy or oesophagogastrectomy, and reconstruction. Between May 1999 and November 2002, forty-four consecutive patients (thirty-eight males and six females; mean age 62, range 30–82) with oesophageal carcinoma (stages I–III), who had undergone radical resection and reconstruction, entered this study (early enteral feeding group; EEF). A historical group of forty-four patients (thirty-seven males and seven females; mean age 64, range 41–79; stages I–III) resected between January 1997 and March 1999 served as control (parenteral feeding group; PF). The duration of both postoperative stay in the Intensive Care Unit (ICU) and the total hospital stay, perioperative complications and the overall mortality were compared. Early enteral feeding was administered over the jejunal line of a Dobhoff tube. It started 6 h postoperatively at a rate of 10 ml/h for 6 h with stepwise increase until total enteral nutrition was achieved on day 6. In the controls oral enteral feeding was begun on day 7. If compared to the PF group, EEF patients recovered faster considering the duration of both stay in the ICU and in the hospital. There was a significant difference in the interval until the first bowel movements. No difference in overall 30 d mortality was identified. A poor nutritional status was a significant prognostic factor for an increased mortality. Early enteral feeding significantly reduces the duration of ICU treatment and total hospital stay in patients who undergo oesophagectomy or oesophagogastrectomy for oesophageal carcinoma. The mortality rate is not affected.

Although the effects of short-term fasting on serum leptin concentrations are known, those resulting from long-lasting modifications of food intake schedule, as during the month of Ramadan, have not yet been extensively studied. Therefore, serum concentrations of leptin were measured around the clock at 4-hourly intervals before the beginning of Ramadan and on the twenty-third day of Ramadan daytime fasting in ten male subjects keeping the same usual activity pattern and general synchronisation in both situations. Time series were analysed with repeated measures ANOVA and Cosinor. No significant changes in amplitude or 24 h mean concentration were seen, but significant shifts of 5 h 30 min in peak and trough serum leptin levels were found on the twenty-third day of Ramadan.

The validity of fruit and vegetable intake estimated by 14 d weighed records, a twenty-seven-item food frequency questionnaire (FFQ) and a 180-item FFQ was investigated using serum carotenoids as the validity criterion. In addition, the method of triads was used to assess the validity of fruit and vegetable intake estimated from the FFQ and serum carotenoids. One hundred Norwegian men completed 14 d weighed records and the 180-item FFQ. Eighty-six of them also completed the twenty-seven item FFQ. The partial correlation coefficients between serum carotenoids and fruit and vegetable intake were slightly higher for the 14 d weighed records than for the two FFQ, but no difference was observed between the 180- and the twenty-seven item FFQ. The strongest correlations were observed between vegetable intake and serum α-carotene. The highest validity coefficients (VC) were observed for vegetable intake estimated from weighed records, the 180-item FFQ, the twenty-seven item FFQ and by the biomarker serum α-carotene, with VC of 0·77, 0·58, 0·51 and 0·67, respectively. In conclusion, the short FFQ gave as valid estimates for fruit and vegetable intake as the long FFQ. Both the estimated partial correlation coefficients and VC suggest that serum α-carotene is the best biomarker for intake of vegetable alone and total intake of fruit and vegetables in this population of Norwegian men, but the biomarkers did not perform any better than the FFQ.

The dietary intake of certain fatty acids might contribute to the development of allergic diseases such as hay fever and asthma. We investigated the association between the concentrations of fifteen fatty acids in serum phospholipids, as a marker of dietary intake and metabolism, and hay fever, allergic sensitisation and total IgE in adults. Data from a population-based cross-sectional study on respiratory health, including the measurement of fatty acids in the serum phospholipids of 740 adults between 20 and 64 years of age, were analysed. Positive associations were found between hay fever and arachidonic acid, and allergic sensitisation and oleic acid. No other fatty acids showed any association with hay fever or allergic sensitisation. Elevated levels of total IgE were not related to fatty acids. Concentrations of long-chain n-3 fatty acids, trans fatty acids or saturated fatty acids in serum phospholipids were not associated with allergic diseases in adults in this study. The present result on the association between hay fever and arachidonic acid is consistent with current hypotheses but warrants further research.

The National School Fruit Scheme (NSFS) provides one free piece of fruit each school day to children, aged 4 to 6 years, attending state schools in England. The aims of the present study were to determine if NSFS was associated with a higher fruit consumption in infant school children (4–6 years old), and to assess whether fruit consumption was higher in junior school children (7–8 years old) who had received free fruit as infants compared with those who had not. The present cross-sectional study involved seventeen schools, eight in the NSFS (study schools) and nine not in the NSFS (control schools). Study and control schools were selected in areas of similar levels of deprivation. All schools were on the outskirts of London in Southeast England. A retrospective 24 h food tick list was given to each pupil in Reception to Year 4 to take home for their parents to complete and return. Response rate was 51 %. Median total fruit consumption (excluding fruit juice) in infants receiving free fruit was 117 g/d compared with 67 g/d in infants not receiving free fruit (P<0·001). Median consumption in juniors who had received free fruit at school as infants did not differ from those who had not (83 g/d v. 86 g/d). The NSFS has increased fruit consumption in infant school children. It does not appear to have longer-term effects in junior school children. If the scheme is to affect dietary habits and improve health in the long term, further interventions will be needed.

Epidemiological surveys suggest that a higher intake of tea may be associated with a lower risk of CHD. There is accumulating evidence that postprandial lipaemia makes a substantial contribution to the incidence of CHD. Our aim was, therefore, to evaluate the effect of tea catechins (major ingredients in green tea) on postprandial lipid responses in human subjects after the consumption of test meals. In a randomized triple-crossover design, nine male subjects with mild or borderline hypertriacylglycerolaemia consumed 10 (control), 224 (moderate dose) and 674 mg (high dose) of the assigned tea catechins three times each along with a standardized light meal consisting of a piece of bread spread with 20 g butter. Plasma lipids were measured in the fasting state and 1, 2, 3, 4 and 6 h after consuming the light meal. Results showed that, compared with the control, moderate and high doses of tea catechins reduced the incremental area under the plasma triacylglycerol curves by 15·1 and 28·7 %, respectively. Next, the rapid elevation of remnant-like particle cholesterol was significantly inhibited by a high dose of tea catechins 2 h after consuming the light meal (P<0·01). In the range of tea catechin dosages, no significant differences were observed in the postprandial responses for plasma total cholesterol or NEFA at any time point. In conclusion, this trial demonstrated that tea catechins attenuated the postprandial increase in plasma triacylglycerol levels following a fat load. These results may provide evidence for one of the possible mechanisms involved in lowering the incidence of CVD, and may prove useful in further studies on the beneficial health effects of tea drinking.

The present study investigated the effect of maternal vitamin E and fatty acid supplementation on lamb antioxidant status. Forty-eight ewes were fed one of four concentrate diets supplemented with a basal (50 mg/kg) or supranutritional (500 mg/kg) level of vitamin E plus a source of either saturated fat (Megalac®; Volac Ltd, Royston, Hertfordshire, UK) or long-chain PUFA (fish oil) from 6 weeks prepartum until 4 weeks postpartum. Blood samples were taken from ewes and lambs at intervals throughout the experiment and, at parturition, muscle, brain and blood samples were obtained from twelve lambs (three per treatment). Colostrum and milk samples were obtained at 12 h and 21 d after parturition, respectively. Supranutritional vitamin E supplementation of the ewe significantly increased concentrations of vitamin E in neonatal lamb tissues although plasma concentrations were undetectable. A significant increase in lamb birth weight resulted from increasing the dietary vitamin E supply to the ewe. Furthermore, maternal plasma, colostrum and milk vitamin E concentrations were increased by vitamin E supplementation, as were lamb plasma concentrations at 14 d of age. Neonatal vitamin E status was not significantly affected by fat source although plasma vitamin E concentrations in both ewes and suckling lambs were reduced by fish oil supplementation of the ewe. Fish oil supplementation reduced vitamin E concentrations in colostrum and milk and the activity of glutathione peroxidase in suckling lambs. The data suggest that the vitamin E status of the neonatal and suckling lamb may be manipulated by vitamin E supplementation of the ewe during pregnancy and lactation.

The effects of dietary isohumulones, the main components accounting for the bitter taste of beer, on lipid metabolism were examined. Young female C57BL/6N mice were fed diets containing isomerized hop extract (IHE), which consists mainly of isohumulones. Administration of IHE with an atherogenic (high-fat and high-cholesterol) diet for 2 weeks resulted in a significant increase in plasma HDL-cholesterol (P<0·01), along with a concomitant reduction in the atherosclerosis index, an increase in liver weight and a decrease in body weight gain in a dose-dependent manner. When animals received IHE with either a cholesterol or a basal diet for 1 week, significant decreases in the liver content of cholesterol (P<0·01) and triacylglycerol (cholesterol diet, P<0·01) were observed. Quantitative analyses of hepatic mRNA levels revealed that IHE administration resulted in up-regulation of mRNA for acyl-CoA oxidase, acyl-CoA synthetase, hydroxymethylglutaryl-CoA synthetase, lipoprotein lipase and fatty acid transport protein, and down-regulation of mRNA for Apo CIII and Apo AI. Administration of purified isohumulones effectively resulted in the same changes as IHE. Administration of fenofibrate, an agonist for PPARα, with a cholesterol diet caused marked hepatomegaly, an increase in plasma HDL-cholesterol, a decrease in hepatic cholesterol content, and alterations in hepatic mRNA levels similar to those observed in mice given IHE. Taken together, these results suggest that the modulation of lipid metabolism observed in mice fed diets containing isohumulones is, at least in part, mediated by activation of PPARα.