Homocysteine has been related to increased risk of CVD. Matrix degradation and inflammation may be involved in this link between hyperhomocysteinaemia and CVD. Recent studies suggest that cystatin C can modulate matrix degradation and inflammation. The present study measured cystatin C at protein (plasma) and mRNA levels (peripheral blood mononuclear cells (PBMC)) in hyperhomocysteinaemic individuals (n 37, female seven and male thirty, aged 20–70 years) before and after B-vitamin supplementation for 3 months in a randomised, placebo-controlled double-blind trial. In a cross-sectional study, seventeen of the hyperhomocysteinaemic subjects were age- and sex-matched to healthy controls (n 17). Our main findings were: (i) as compared with controls, hyperhomocysteinaemic subjects tended to have higher plasma concentrations of cystatin C and lower mRNA levels of cystatin C in PBMC; (ii) compared with placebo, treatment of hyperhomocysteinaemic individuals with B-vitamins significantly increased plasma levels of cystatin C and mRNA levels of cystatin C in PBMC; (iii) while plasma levels of cystatin C were positively correlated with plasma levels of TNF receptor-1, mRNA levels of cystatin C in PBMC were inversely correlated with this TNF parameter. Taken together, our findings suggest that disturbed cystatin C levels may be a characteristic of hyperhomocysteinaemic individuals, potentially related to low-grade systemic inflammation in hyperhomocysteinaemic subjects, and that B-vitamins may modulate cystatin C levels in these individuals.

Necrotising enterocolitis (NEC) is a devastating gastrointestinal disease of prematurity that typically develops after the administration of infant formula, suggesting a link between nutritional components and disease development. One of the most significant complications that develops in patients with NEC is severe lung injury. We have previously shown that the administration of a nutritional formula that is enriched in pre-digested Triacylglyceride that do not require lipase action can significantly reduce the severity of NEC in a mouse model. We now hypothesise that this ‘pre-digested fat (PDF) system’ may reduce NEC-associated lung injury. In support of this hypothesis, we now show that rearing newborn mice on a nutritional formula based on the ‘PDF system’ promotes lung development, as evidenced by increased tight junctions and surfactant protein expression. Mice that were administered this ‘PDF system’ were significantly less vulnerable to the development of NEC-induced lung inflammation, and the administration of the ‘PDF system’ conferred lung protection. In seeking to define the mechanisms involved, the administration of the ‘PDF system’ significantly enhanced lung maturation and reduced the production of reactive oxygen species (ROS). These findings suggest that the PDF system protects the development of NEC-induced lung injury through effects on lung maturation and reduced ROS in the lung and also increases lung maturation in non-NEC mice.

Fe deficiency is a public-health problem worldwide, and effective measures for preventing Fe deficiency are needed. The aim of the present study was to determine the bioavailability of Fe in cocoa using the Hb regeneration efficiency (HRE) method. Thirty-five F344/N male weanling rats were fed a low-Fe diet for 4 weeks to deplete body Fe stores. Then, four groups of seven animals each were repleted for 20 d using a modified AIN-93G diet fortified with ferrous sulphate, ferric citrate or two brands of cocoa powder to provide a total dietary Fe concentration of 20 mg/kg. As a negative control, seven rats were maintained on the low-Fe diet. The HRE were 0·733, 0·350, 0·357 and 0·336 for ferrous sulphate, ferric citrate and the two brands of cocoa powder, respectively. The relative biological values (RBV), defined as the ratio of the sample HRE to that of ferrous sulphate, were 0·478, 0·488 and 0·459 for ferric citrate and the two brands of cocoa powder, respectively. The Fe bioavailability of cocoa was significantly less than that of ferrous sulphate and was similar to that of ferric citrate. The difference in Fe bioavailability between the two brands of cocoa powder was negligible. When the negative control was used to correct the data, estimates of the RBV derived from Hb gain were similar to those derived from the HRE. These results suggest that cocoa is a significant source of moderately bioavailable Fe.

The first investigation of dietary intake in the Mediterranean region was undertaken at the initiative of the government of Greece in 1948. Plant foods (cereals, pulses, nuts, potatoes, vegetables and fruits) accounted for 61 % of total energy intake (TEI), animal foods (meat, eggs, fish and dairy products) for 7 % of TEI and olive oil was the main oil used. In 1950s, Ancel Keys undertook studies in USA, Italy, Spain, England, Japan, Australia and Canada leading him to hypothesise that a link could exist between diet, plasma cholesterol and CHD. Between 1958 and 1964, Keys and co-workers carried out the Seven Countries Study, which enrolled men aged 40–59 years in one of sixteen cohorts from seven countries (Finland, Greece, Italy, Yugoslavia, Japan, USA and Italy). After 15-, 25- and 50-year follow-up, a strong positive relation was observed between saturated fat intake and CHD mortality, and a negative one with Mediterranean Dietary Index. In 1975, Keys and his wife published a book entitled: ‘How to eat well and stay well. The Mediterranean way’, which popularised Mediterranean Diet (MedDiet). After 45-year follow-up, longevity without CHD death was 12·9 years higher in Crete than in Finland. Protecting effect of MedDiet towards CHD incidence and risk is now confirmed by Prevencion con Dieta Mediterranea study and by cohorts’ studies gathered in several recent meta-analyses. MedDiet is sustainable and recognised by UNESCO as an intangible cultural heritage, which is the most beautiful homage that can be paid to Ancel Keys and all his co-workers.

The consumption of nuts and extra-virgin olive oil has been associated with suppression of inflammatory pathways that contribute to atherosclerosis, but its role on the modulation of the inflammatory profile in patients with established coronary artery disease (CAD) is unclear. The aim of this study was to evaluate the effects of adding pecan nuts or extra-virgin olive oil to a healthy diet on inflammatory markers in patients with stable CAD. In this randomised clinical trial, 204 patients were enrolled to three study groups: sixty seven to control group (CG: healthy diet), sixty eight to pecan nuts group (PNG: 30 g/d of pecans + healthy diet) and sixty nine to extra-virgin olive oil group (OOG: 30 ml/d of extra-virgin olive oil + healthy diet). High-sensitivity C-reactive protein (hs-CRP, in mg/l), fibrinogen (mg/dl), IL 2, 4, 6, 10 (pg/ml) and interferon-γ (IFN-γ, in pg/ml), IL-6/IL-10, IL-2/IL-4 and IFN-/γIL-4 ratios were evaluated at baseline and after the follow-up (12 weeks). As main results, after adjustment for sex, statin used and relative body weight variation, there were no differences between groups regarding inflammatory markers at the end of the study. IL-6 levels (primary outcome) were reduced in 12 weeks when compared with baseline in all study groups (CG: difference: −0·593 (se = 0·159) pg/dL; PNG: difference: −0·335 (se = 0·143) pg/dl; OOG: IL-6 difference: −0·325 (se = 0·143) pg/dl). In conclusion, there was no significant effect of including pecan nuts or extra virgin olive oil to a healthy diet on inflammatory markers in individuals with CAD.

1. Fatty liver and kidney syndrome (FLKS) was induced in young broiler chickens by giving them a diet composed principally of wheat and meat meal.

2. FLKS resulted in reduced growth and increased liver weight; fasting for 18 h increased mortality, liver lipid and the specific activity of hepatic ATP-citrate lyase compared with birds fed on a commercial diet. The specific activities of hepatic fructose-l,6-diphosphate-l-phosphohydrolase and pyruvate carboxylase were reduced in birds suffering from FLKS and fasted for 18 h.

3. Feeding of the FLKS-inducing diet supplemented with 150 g animal tallow/kg for 54 h considerably reduced mortality while restoring liver composition and enzyme activities towards those observed in birds fed a commercial diet. Investigations indicated that the glycerol component of the fat was not responsible for the observed responses.

4. The present results suggest that in FLKS insufficiencies of biotin are induced in specific enzyme systems, but the syndrome may be alleviated without the use of supplementary biotin.

5. The evidence indicates that, when stressed, birds affected by FLKS die from the hypoglycaemia occurring as a result of a reduced capacity for gluconeogenesis.

The effect of abomasal infusion of glucose (120 kJ/d per kg body weight (BW)0·75, 758 mmol/d) on urea production, plasma alanine-N flux rate and the conversion of alanine-N to urea was studied in sheep offered a low-N diet at limited energy intake (500 kJ/d per kg BW0·75), based on hay and grass pellets. Glucose provision reduced urinary N (P = 0·040) and urea (P = 0·009) elimination but this was offset by poorer N digestibility. Urea-N production was significantly reduced (822 v. 619 mmol/d, P = 0·024) by glucose while plasma alanine-N flux rate was elevated (295 v. 342 mmol/d, P = 0·011). The quantity of urea-N derived from alanine tended to be decreased by glucose (127 v. 95 mmol/d) but the fraction of urea production from alanine was unaltered (15 %). Plasma urea and alanine concentrations (plus those of the branched chain amino acids) decreased in response to exogenous glucose, an effect probably related to enhanced anabolic usage of amino acids and lowered urea production.

The optimal dosage of ornithine α-ketoglutarate (OKG) for repleting tissue glutamine (Gln) concentrations and maintaining N homeostasis after injury is unknown. We set out to perform ‘dose-ranging’ of OKG supplementation after an endotoxaemic challenge. Sixty-one male Wistar rats were injected with 3 mg lipopolysaccharide (LPS) from Escherichia coli/kg (n 50) or saline vehicle (9 g NaCl/l; controls n 11). After a 24 h fast, survivors were fed by gavage for 48 h with a polymeric standard diet (879 kJ/kg per d and 1·18 g N/kg per d) supplemented with non-essential amino acids (control, n 11; LPS-OKG-0·0, n 9), or with 0·5 g OKG/kg per d (LPS-OKG-0·5, n 12), 1·5 OKG/kg per d (LPS-OKG-1.5, n 11) or 4·5 g OKG/kg per d (LPS-OKG-4·5, n 10). The diets for all groups were made isonitrogenous with the LPS-OKG-4·5 diet by adding an appropriate amount of non-essential amino acids. Rats were killed on day 3 for blood and tissue sampling (muscle, jejunum mucosa, liver). Urine was collected daily for 3-methylhistidine and total N assays. The OKG dose was correlated with Gln concentrations in every tissue and with cumulative N balance (Spearman test, P<0·01). 3-Methylhistidine excretion was increased in endotoxaemic groups compared with controls (ANOVA, P<0·05) except in the LPS-OKG-4·5 group. Only the LPS-OKG-4·5 group achieved a positive post-injury N balance (t test, P<0·05). In conclusion, OKG exerted a dose-dependent effect on tissue Gln concentration and N balance, but only the highest dosage counteracted myofibrillar hypercatabolism and caused a positive N balance.

1. The disaccharide lactulose (galactosyl-β-1,4-fructose) was poorly absorbed from rat small intestine in vitro and human mouth in vivo.

2. These results confirm indirect clinical evidence of poor absorption from the intestine.

3. The presence of calcium ions, or absence of sodium ions, had no effect on lactulose absorption from the buccal cavity.

4. The presence of ouabain, or absence of Na+, did not decrease the absorption of lactulose from small intestine.

5. It is thought that the mode of transport, in both instances, is by passive diffusion with the concentration gradient.

1. Twenty pigs were reared to a weight of 90 kg on various dietary regimens so that their lipid content varied from 16 to 38%;. They were injected intravenously with ~ 400 μCi of 42KCl. The animals were killed ~ 28 h after injection and their contents of water, lipid, sodium and potassium were determined.

The specific activity of plasma reached equilibrium 10–12 h after injection, that of urine not until 20 h after.

Losses of activity in urine, faeces and gut contents in the 24 h after injection amounted to 3·1, 0·3 and 1·5% of the injected dose, respectively.

Total body K was more accurately estimated from urine specific activity than from plasma specific activity.

Exchangeable K, estimated from urine specific activity Ke(u), was highly correlated with the weight of fat-free tissue (r = 0·93), the residual standard deviation of the regression being 1·8 kg fat-free tissue.

Percentage extractable fat was equally well correlated with Ke(u) (r = −0·92).

Few studies have examined the association between coffee consumption and muscle mass; their results are conflicting. Therefore, we examined the association between coffee consumption and low muscle mass prevalence. We also performed an exploratory investigation of the potential effect modification by demographic, health status-related and physical activity-related covariates. This cross-sectional study included 2085 adults aged 40–87 years. The frequency of coffee consumption was assessed using a self-administered questionnaire. Muscle mass was assessed as appendicular skeletal muscle mass/height2 using a multifrequency bioelectrical impedance analyser. We defined low muscle mass using cut-offs recommended by the Asian Working Group for Sarcopenia. Multivariable-adjusted OR for low muscle mass prevalence were estimated using a logistic regression model. The prevalence of low muscle mass was 5·4 % (n 113). Compared with the lowest coffee consumption group (< 1 cup/week), the multivariable-adjusted OR (95 % CI) of low muscle mass prevalence were 0·62 (0·30, 1·29) for 1–3 cups/week, 0·53 (0·29, 0·96) for 4–6 cups/week or 1 cup/d and 0·28 (0·15, 0·53) for ≥ 2 cups/d (P for trend < 0·001). There were no significant interactions among the various covariates after Bonferroni correction. In conclusion, coffee consumption may be inversely associated with low muscle mass prevalence.

1.It has been postulated that the vitamin B12-binding protein in the sows' milk may facilitate the intestinal absorption of vitamin B12 in the piglet. This implies that the binder is not rapidly and completely degraded by gut proteases, and the present experiments were devised to test this assumption.

2. Piglets aged 7 and 28 d were given a test feed of sows' milk whey both with and without cyano[3H]cobalamin to saturate the binder, and with 14C-labeHed polyethylene glycol (PEG) as a marker. Control piglets were given a milk substitute containing no vitamin B12-binder. At 80 min after the test meal the piglets were killed and the contents of the stomach and small intestine were removed for analysis.

3. Recovery of [14C]PEG, mainly from the middle and distal portions of the small intestine, ranged from 49 to 78%. Apparent loss of vitamin B12-binder from the intestine was calculated from the ratio, PEG: binder. In both 7- and 28-d-old piglets, the content of saturated binder in the middle and lower small intestine was about 20% depleted relative to that in the test feed. With the unsaturated binder the loss was more variable and generally greater, at about 50%.

4. At slaughter the stomach was nearly empty and the high unsaturated binding capacity of the contents was attributed to endogenous binders. However, in the middle and lower small intestine, the binding capacity was derived mainly from sows' milk given with the test meal. No free cyano[3H]cobalamin was found in the intestinal contents of piglets given sows' milk to which enough cyano[3H]cobalamin had been added to saturate the binder.

5. There was no change in the molecular weight of the vitamin B12-binder during its passage down the intestine, as judged by its behaviour on filtration in Sephadex gel G-150.

6. The results indicate that a high proportion of saturated binder, and a smaller proportion of unsaturated binder, survived unchanged in the intestine of piglets at both ages.

Carotenoids are increasingly being implicated to have an important role in brain and eye development. This study aimed to quantify the content and profile of carotenoids in human breast milk, maternal plasma and neonatal umbilical cord plasma in Chengdu, an urban area in Southwest China. In this study, fifty-four healthy mothers were enrolled. Maternal blood, umbilical cord blood, colostrum, transitional milk and mature milk were collected. Concentrations of carotenoids (lutein, zeaxanthin, β-cryptoxanthin, β-carotene and lycopene) were analysed by HPLC. We found that carotenoid concentrations decreased from colostrum to mature milk. Hydrocarbon carotenoids with weaker polarity decreased more than the polar carotenoids. Lycopene concentrations dropped by 99 %, β-carotene by 92 %, β-cryptoxanthin by 83 %, lutein by 32 % and zeaxanthin by 22 %. Lycopene and β-carotene accounted for 70 % of the total carotenoids in colostrum, and lutein predominated amongst carotenoids in transitional milk and mature milk (51–55 %). Carotenoid concentrations in maternal plasma were much higher than that in cord plasma. Lutein predominated in cord plasma. The concentrations of all carotenoids in maternal plasma were correlated with those of cord plasma and human milk. These results are consistent with selective transport mechanisms in the mammary gland related to the polarity of carotenoids, and each carotenoid has its own implications, which may have different priorities in the early life development of infants. These findings may help guide dietary recommendations for carotenoid inclusion in infant formulas.

The declining inflammatory immune competence of acute (i.e. wasting) pre-pubescent protein–energy malnutrition has been regarded as reflecting an unregulated immunological disintegration. Recent evidence, however, suggests that malnutrition stimulates a regulated immunological reconfiguration to achieve a non-inflammatory form of competence, perhaps offering protection against autoimmune reactions – the ‘Tolerance Model’. Our objective was to determine the influence of acute pre-pubescent malnutrition on the expression of genes critical to tolerogenic regulation. Male and female C57BL/6J mice, initially 19 d old, consumed a complete purified diet either ad libitum (age-matched controls) or in restricted daily quantities (mimicking marasmus), or consumed an isoenergetic low-protein diet ad libitum (mimicking incipient kwashiorkor) for 14 d (six animals per dietary group). Gene expression in the spleen, typically an inflammatory organ, and in the small intestine, a site designed for non-inflammatory defence, was assessed by real-time quantitative RT-PCR, and normalised to β-actin. In the spleen of the malnourished groups, both IL-10 and transforming growth factor-β1 mRNA expression increased compared with controls (P < 0·05), whereas mRNA expression of IL-12p40 decreased (P < 0·05). Conversely, malnutrition exerted no influence on the expression of mRNA for these cytokines in the small intestine (P>0·05). Moreover, forkhead box P3 mRNA expression, indicative of cell-based tolerogenic potential, was sustained in both the spleen and intestine of the malnourished groups (P>0·05). Thus, despite limited supplies of energy and substrates, the spleen shifted towards a non-inflammatory character and the intestine was sustained in this mode in advanced pre-pubescent weight loss. These findings provide the first support for the Tolerance Model at the level of mRNA transcript expression.

From days 4–104 of pregnancy, adolescent sheep, weighing 43·7 (SE 0·87)kg were offered a complete diet at two different intakes (approximately 5 or 15 kg/week) designed to meet slightly, or well above, maternal maintenance requirements. The fetal and maternal muscles were taken on day 104 of pregnancy and analysed for total DNA, RNA and protein. Ewes offered a high intake to promote rapid maternal weight gain, weighed more (76·5 (SE 4·5) v. 50·0 (SE 1·7) kg) and had muscles with a greater fresh weight, whilst their fetuses had smaller muscles, than those fed at a lower intake. Plantaris muscle of the ewes fed at the high intake contained more RNA and protein; again the opposite situation was found in the fetal muscle. On the higher maternal intakes, the DNA, RNA and protein contents of the fetal plantaris muscle were less than in fetuses of ewes fed at the lower intake. To investigate the possible mechanisms involved in this decrease in fetal muscle mass, cytosolic and membrane-associated muscle proteins were subjected to Western immunoblotting with antibodies to nine isoforms of protein kinase C (PKC), a family of enzymes known to play an important role in cell growth. Five PKC isoforms (α, ε, θ, μ and ζ) were identified in fetal muscle. One of these, PKC-α, was located predominantly in the cytosolic compartment in the smaller fetuses of the ewes fed at a high plane of nutrition, but was present to a greater extent in the membranes of the more rapidly growing fetuses of the ewes fed at the lower intake. This was the only isoform to demonstrate nutritionally related changes in its subcellular compartmentation suggesting that it may mediate some aspects of the change in fetal growth rate.

Cryptomys hottentotus has no access to dietary or endogenous sources of cholecalciferol (D3). Serum concentrations of calcifediol (25(OH)D3) were undetectable (< 5 ng/ml) and calcitriol (1,25(OH)2D3), although detectable in plasma, was at a low concentration (31.40) (SEM 3.06 pg/ml). Despite their naturally impoverished vitamin D status, these animals exhibited highly efficient mineral absorption processes (Ca apparent fractional absorption efficiency, AFA (%) 95.33 (SEM 1.36); inorganic P (Pi) AFA (%) 93.49 (SEM 0.81)). Furthermore, plasma mineral content was tightly regulated (Ca 2.57 (SEM 0.08) mmol/l; Mg 1.23 (SEM 0.05) mmol/l; Pi 2.12 (SEM 0.15) mmol/1). Mode of uptake in C. hottentotus was unlike that in other D3-replete mammals. First, passive (rather than active) uptake occurred in the traditional site of active Ca absorption (with serosal: mucosal (S:M) ratios in the duodenum of 1.32 (SEM 0.13)), and the only site of active Ca uptake was the hindgut (caecum S:M 3.35 (SEM 0.46); proximal colon S:M 2.13 (SEM 0.30)). Despite the presence of active uptake in these hindgut regions, their overall contribution to the daily rate of mineral absorption was small (9.53 (SEM 1.27)%). These underground inhabitants rely upon highly efficient, passive mineral uptake. This is adequate to meet their mineral requirements and maintain mineral homeostasis in the absence of vitamin D.

Folates, an essential component (important B vitamin) in the human diet, are involved in many metabolic pathways, mainly in carbon transfer reactions such as purine and pyrimidine biosynthesis and amino acid interconversions. Deficiency of this micronutrient leads to the disruption of folate-dependent metabolic pathways that lead to the development of clinical abnormalities ranging from anaemia to growth retardation. Folate deficiency due to alcohol ingestion is quite common, primarily due to malabsorption. The present study dealt with the mechanistic insights of folate malabsorption in colonic basolateral membrane (BLM). Wistar rats (n 12) were fed 1 g/kg body weight per d ethanol (20 %) solution orally for 3 months and folate transport was studied in the isolated colonic BLM. The folate exit across colon BLM shows characteristics of carrier-mediated process with the major involvement of reduced folate carrier (RFC). The chronic ethanol ingestion decreased the uptake by decreasing the affinity by 46 % (P < 0·01) and the number of transport molecules by 43 % (P < 0·001) at the colon BLM. The decreased uptake was associated with down-regulation of proton-coupled folate transporter (PCFT) and RFC expression at mRNA and protein levels. The extent of decrease was 44 % (P < 0·01) and 24 % (P < 0·05) for PCFT and 23 % (P < 0·01) and 57 % (P < 0·01) for RFC at mRNA and protein levels, respectively. Moreover, folate transporters were associated with lipid rafts (LR) of colon BLM, and chronic alcoholism decreased the association of these transporters with LR.

Misalignment of day/night and feeding rhythms has been shown to increase fat deposition and the risk for metabolic disorders in humans and rodents. In most studies, however, food intake and intake patterns are not controlled. We studied the effects of circadian misalignment on energy expenditure in pigs while controlling for food intake as well as intake patterns. Twelve groups of five male pigs were housed in respiration chambers and fed either during the day (10.00–18.00 hours; DF) or night (22.00–06.00 hours; NF), bihourly the same sequential meals, representing 15, 10, 25, 30 and 20 % of the daily allowance. Paired feeding was applied to ensure equal gross energy intake between treatments. Apparent total tract digestibility, energy balances and heat partitioning were measured and analysed using a mixed linear model. Apparent total tract energy and DM digestibility tended to be lower for NF-pigs than DF-pigs (P < 0·10). Heat production was 3 % lower for NF-pigs than DF-pigs (P < 0·026), increasing fat retention by 7 % in NF-pigs (P = 0·050). NF-pigs were less active than DF-pigs during the feeding period, but more active during the fasting period. RMR was greater for DF-pigs than NF-pigs during the fasting period. Methane production was 30 % greater in NF-pigs than DF-pigs (P < 0·001). In conclusion, circadian misalignment has little effect on nutrient digestion, but alters nutrient partitioning, ultimately increasing fat deposition. The causality of the association between circadian misalignment and methane production rates remains to be investigated.