In rats, in contrast with human subjects who develop megaloblastic anaemia due to vitamin B12 deficiency, haematological abnormalities with anaemia were not observed under normoxic conditions even though plasma vitamin B12 concentration was reduced to <15 % of a normal concentration by depleting dietary vitamin B12. To elucidate whether erythropoiesis was affected by vitamin B12 deficiency in rats, these vitamin B12-deficient rats were exposed to hypoxia (10·5 % O2) to stimulate erythropoiesis. In the vitamin B12-sufficient control rats, erythrocyte count was significantly (P<0·05) increased 1 week after starting the hypoxic exposure. However, the hypoxia-induced erythropoiesis was affected by vitamin B12 deficiency, and no significant increase in the erythrocyte count was observed even after 6-week exposure to hypoxia in the vitamin B12-deficient rats. In the vitamin B12-deficient rats in hypoxia, erythrocytes became abnormally enlarged, and haemoglobin concentration in peripheral blood was increased in proportion to the increase of mean corpuscular volume. However, the level of the increase in the haemoglobin concentration was significantly (P<0·05) lower in the vitamin B12-deficient rats compared with that in the -sufficient controls. In addition, in the vitamin B12-deficient rats, in contrast to the -sufficient rats, serum erythropoietin concentration was not normalized even after 6-week exposure to hypoxia. These results indicate that a megaloblastic anaemia-like symptom is induced when the vitamin B12-deficient rats are exposed to hypoxia.

Tropical livestock is often maintained on roughage-based diets deficient in N, and therefore requires supplementation with protein-rich substrates to achieve reasonable production levels. The optimum inclusion rate of a potential supplement is usually determined by in vivo feeding trials or by in vitro incubation of the diet components to estimate the feed value of the complete diet. The present work simulates a supplementation experiment in vitro, by incubating a pure roughage (barley straw), a pure supplement (Sesbania pachycarpa leaves) and mixtures of the two, with increasing inclusion levels of the supplement, in a short-term batch incubation system. Fermentation kinetics were followed by the release of fermentation endproducts (gas and short-chain fatty acids). Microbial biomass was estimated using ribosomal (r) RNA as internal marker for bacteria and eukaryotes separately. Cell-wall-degrading subpopulations were quantified by hybridisation with taxon-specific oligonucleotide probes targeting Chytridiomycetes, Fibrobacter spp., Ruminococcus albus and R. flavefaciens. Carboxymethylcellulase (CMCase) was assayed as an indicator for cell-wall-degrading activity. The addition of S. pachycarpa leaves stimulated fermentation in all cases. Gas production, and especially rRNA concentration, showed clear maxima at 40 % S. pachycarpa inclusion, rates that significantly exceeded the values interpolated from the incubations of the pure substrates. Short-chain fatty acid yield changed only slightly, but in the same way. The analysis of the microbial population structure showed that the positive effects were mainly mediated through enhanced growth of Ruminococcus spp. Increasing proportions of S. pachycarpa leaves in the diet led to a drastic decline in the total eukaryotic population. This points to a defaunation, which may also have added to the positive effects. The eukaryotic subpopulation of the rumen fungi were affected to a lesser degree. Although the cell-wall-degrading organisms showed positive responses to the supplementation, the CMCase activity was not affected significantly by the supplementation. The present work shows that it is possible to predict optimum inclusion levels for a new feed supplement in vitro and thus reduce in vivo experiments. It was also demonstrated that true supplementation effects occur particularly for the microbial biomass production, which is the primary source of amino acids for the ruminant animal. The analysis of microbial population structure in context with conventional metabolic measurements adds valuable information to interpret the observed effects on production-related variables.

The accepted biological potencies of vitamin E (United States Phamacopeia, 1985) for 1 mg all-rac-α-tocopheryl acetate (synthetic form) is 1·00 IU and that of 1 mg (RRR)-α-tocopheryl acetate (natural form) is 1·36 IU. In the present study, a stable isotopic (2H) technique was employed to evaluate the bioavailability of natural v. synthetic forms of vitamin E and to determine whether the potency of the forms is the stated relationship of 1·36:1·00 (RRR)-α-tocopheryl acetate:all-rac-α-tocopheryl acetate. Sixty female in-bred guinea pigs received either 40 or 80 mg vitamin E/kg diet with equal levels of (RRR)-α-tocopheryl acetate and all-rac-α-tocopheryl acetate throughout gestation and lactation. At late-term pregnancy (day 50 or 60) and during early lactation, dams and their corresponding fetuses or neonates were killed and various tissues collected for subsequent α-tocopherol analysis. Vitamin E analysis of fetal and neonatal tissues indicated a substantial transfer of 2H-labelled α-tocopherol across the placenta and through the mammary gland. Total α-tocopherol concentrations were significantly influenced by tissue type and dose level, but not by stage of gestation or lactation. The relative bioavailability (d3:d6) across fetal and neonatal tissues was on average 1·81:1·00, with a range from 1·62:1·00 to 2·01:1·00. Maternal tissues had a mean ratio of 1·77:1·00. A higher relative bioavailability (P≤0·05) was observed with natural compared with synthetic α-tocopherol as shown by a higher d3:d6 ratio in all tissues examined. Vitamin E was highest in colostrum on day 2 then declined through to day 5. Results from this present experiment further question the accepted biological potencies of natural:synthetic α-tocopheryl acetate of 1·36:1·00.

Consumption of isoflavone-rich soyabean protein is reported to reduce total and LDL-cholesterol, but the specific components responsible are undetermined. In a previous crossover trial we showed that purified isoflavones, derived from red clover (Trifolium pratense), raised HDL3-cholesterol in premenopausal women; however, these findings were inconclusive due to period and carryover effects. In an attempt to overcome this problem, we utilised a parallel study designed to re-examine the effects of purified isoflavones on plasma lipoproteins and markers of insulin resistance in premenopausal women. Twenty-five healthy premenopausal women participated in a double-blind, randomised, parallel study. The treatment group (n 12) consumed a placebo for the first menstrual cycle and an isoflavone supplement (86 mg/d, derived from red clover) for three cycles, while the placebo group (n 13) consumed a placebo supplement for four menstrual cycles. Blood samples were collected weekly during cycles 1, 3 and 4. Supplementation with isoflavones resulted in a 15-fold increase in urinary isoflavone excretion (P<0·0001). There were no significant effects on total cholesterol, LDL- and HDL-cholesterol, HDL subfractions, triacylglycerol, lipoprotein(a), glucose or insulin concentrations. Our present results indicate that purified isoflavones derived from red clover have no effect on cholesterol homeostasis or insulin resistance in premenopausal women, a group which is at low risk of CHD.

A cross-sectional study of 132 adults attending an HIV clinic in Cape Town, South Africa, was conducted to determine predictors of low plasma vitamin A and Zn levels. No patients were on antiretroviral therapy. The possible confounding effect of the acute-phase response was controlled by including C-reactive protein levels in multivariate analysis and by excluding active opportunistic infections. Retinol levels were low (<1·05 μmol/l) in 39 % of patients with early disease (WHO clinical stages I and II) compared with 48 and 79 % of patients with WHO stage III and IV respectively (P<0·01). Plasma Zn levels were low (<10·7 μmol/l) in 20 % of patients with early disease v. 36 and 45 % with stage III and IV disease respectively (P<0·05). C-reactive protein levels were normal in 63 % of subjects. Weak, positive associations were found between CD4+ lymphocyte count and plasma levels of retinol (r 0·27; 95 % CI 0·1, 0·43) and Zn (r 0·31; 95 % CI 0·25, 0·46). Multivariate analysis showed the following independent predictors of low retinol levels: WHO stage IV (odds ratio 3·4; 95 % CI 2·1, 5·7) and body weight (odds ratio per 5 kg decrease 1·15; 95 % CI, 1·08, 1·25), while only body weight was significantly associated with low Zn levels (OR per 5 kg decrease 1·19; 95 % CI 1·09, 1·30). CD4+ lymphocyte count <200/μl was not significantly associated with either low retinol or Zn levels. In resource-poor settings, simple clinical features (advanced disease and/or weight loss) are associated with lowered blood concentrations of vitamin A and/or Zn. The clinical significance of low plasma retinol and/or Zn levels is unclear and more research is required to establish the role of multiple micronutrient intervention strategies in HIV disease.

Low n-3 polyunsaturated fatty acid (PUFA) status may be associated with neuro-degenerative disorders, in particular Alzheimer's disease, which has been associated with poor dietary fish or n-3 PUFA intake, and low docosahexaenoic acid (DHA) status. The present case–control study used an established biomarker of n-3 PUFA intake (serum cholesteryl ester-fatty acid composition) to determine n-3 PUFA status in patients with Alzheimer's disease, who were free-living in the community. All cases fulfilled the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer's Disease and Related Disorders Association criteria for Alzheimer's disease. Detailed neuropsychological testing and neuroimaging established the diagnosis in all cases. The subjects (119 females and twenty-nine males) aged 76·5 (SD 6·6) YEARS HAD A CLINICAL DEMENTIA RATING (CDR) OF 1 (sd 0·62) and a mini mental state examination (MMSE) score of 19·5 (sd 4·8). The control subjects (thirty-six females and nine males) aged 70 (sd 6·0) years were not cognitively impaired (defined as MMSE score <24): they had a mean MMSE score of 28·9 (sd 1·1). Serum cholesteryl ester-eicosapentaenoic acid and DHA levels were significantly lower (P<0·05 and P<0·001 respectively) in all MMSE score quartiles of patients with Alzheimer's disease compared with control values. Serum cholesteryl ester-DHA levels were progressively reduced with severity of clinical dementia. DHA levels did not differ in patients with Alzheimer's disease across age quartiles: all were consistently lower than in control subjects. Step-wise multiple regression analysis showed that cholesteryl ester-DHA and total saturated fatty acid levels were the important determinants of MMSE score and CDR. It remains to be determined whether low DHA status in Alzheimer's disease is a casual factor in the pathogenesis and progression of Alzheimer's disease.

The objective of the present experiment was to examine the effect of substituting different quantities of soyabean protein for casein on renal function and lipid metabolism in rats with chronic renal failure induced by a five-sixths nephrectomy. Experimental animals were subjected to a nephrectomy and fed either casein or soyabean protein (200 or 100 g/kg diet). The diets were isoenergetic with identical fat, Na, K and P contents. Rats ingesting 200 g casein/kg diet showed a significantly (P<0·05) accelerated course of chronic renal failure, while the soyabean-protein groups showed retarded progression of the experimentally induced renal disease and hypercholesterolaemic effects. Rats in the low-soyabean-protein diet (100 g/kg) also demonstrated increased serum albumin and decreased serum triacylglycerol, total cholesterol concentrations and blood urea-N; however, the low-casein diet significantly (P<0·05) increased serum triacylglycerol. Results of the present study show that the replacement of casein by soyabean protein was related to the rate of progression of renal failure and improvement in lipid profiles in serum of five-sixths nephrectomized rats.

A whole-body radioassay procedure was used to assess the retention and apparent absorption by rats of Cd in kernels of durum wheat (Triticum turgidum L. var. durum) harvested from plants grown hydroponically in 109Cd-labelled nutrient solution. Wholegrain wheat, containing 5 μmol Cd (570 μg)/kg dry weight labelled intrinsically with 109Cd, was incorporated into test meals fed to rats that had been maintained on diets containing marginally adequate, adequate or surplus levels of Zn (0·12 mmol (8 mg), 0·43 mmol (28 mg) or 1·55 mmol (101 mg) Zn/kg respectively), and either 0 or 50 g durum wheat/kg. Regardless of diet, all rats consumed about 99 % of the test meal offered. In rats fed diets without wheat, initial Cd absorption averaged 7·7, 4·6 and 2·4 % of the dose when the diet contained 0·12 mmol (8 mg), 0·43 mmol (28 mg) or 1·55 mmol (101 mg) Zn/kg diet respectively. In rats fed wheat-containing diets, initial Cd absorption averaged 3·8 and 2·6 % of the dose when dietary Zn concentration was 0·12 mmol (8 mg) and 0·43 mmol (28 mg)/kg diet respectively. The amount of Cd retained in the body at 15 d postprandial was <2 % of the dose in all rats, and decreased as Zn in the diet increased. Even at 15 d postprandial, 32 to 44 % of the Cd retained in the body was still in the gastrointestinal tract. The results show that: (1) the bioavailability to rats of Cd in wholegrain durum wheat was depressed when wholegrain wheat was part of the regular diet; (2) increased intake of dietary Zn lowered Cd absorption and retention; (3) retention of Cd in the body at 15 d postprandial from diets containing adequate Zn was <1·3 %.

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.

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.

Ageing is a multifactorial process involving decreased antioxidant defences and immune functions. n-3 Polyunsaturated fatty acids have been associated with human health benefits, especially against inflammatory and autoimmune diseases. However, their immunomodulatory effects were usually observed with high dosages (>2 g/d) known to increase lipid peroxidation. In contrast, very low doses, that may prevent lipid peroxidation, might affect the immune system differently. To study the latter hypothesis further, we investigated whether the supplementation of healthy elderly people with very low doses of marine oil (MO), a docosahexaenoate (DHA)- and eicosapentaenoate (EPA)-rich triacylglycerol, was able to affect lymphocyte proliferation and biochemical markers known to be altered with age. In a randomized, double-blind design, twenty healthy elderly subjects were assigned to a placebo group (600 mg sunflower oil/d) or to a group consuming 600 mg MO/d providing 150 mg DHA + 30 mg (EPA) for 6 weeks. At day 42, the proliferative responses of lymphocytes to several mitogens were significantly (P<0·01) decreased in the MO group compared with control values. This was accompanied by a slight lowering of their cytosolic cyclic nucleotide phosphodiesterase (PDE) activity, a marked and significant (P<0·05) increase of their particulate PDE activity (+56–57 %) and a slight but significant (P<0·05) increase in cyclic nucleotide intracellular levels. At the same time, the glutathione peroxidase activity was markedly and significantly (P<0·01) depressed in the MO group. None of these modifications could be seen in the placebo group. Collectively, these results demonstrate that even very low doses of n-3 fatty acids are sufficient to affect the immune responses of elderly subjects.

The aim of the present study was to obtain serial values of O2 consumption (VO2), CO2 production (VCO2) and energy expenditure (EE) in healthy but extremely-low-birth-weight infants (birth weight <1000 g), during the first 5 weeks after birth. A total of seventeen spontaneously breathing and appropriate-for-gestational-age (birth weight and body length above the 10th and below the 90th percentile) preterm infants with gestational age 25–28 weeks and birth weight 590–990 g were enrolled in the study. Calorimetry was performed using an open-circuit calorimeter on days 6, 12, 18, 24, 30 and 36 of postnatal life. During the 5 weeks of observation, VO2 increased from 4·7 (SD 0·5) TO 9·1 (sd 1·0) ml/kg per min, VCO2 from 4·5 (sd 0·4) to 8·3 (SD 0·6) ml/kg per min and EE from 115 (sd 12) to 310 (sd 71) kJ/kg per d. The energy intake was always higher than EE, even at days 6 and 12. The RER decreased from 0·99 (sd 0·09) at day 12 to 0·91 (SD 0·05) at day 30. On all study days, there were highly significant positive correlations between energy intake and weight gain, EE and weight gain, and EE and energy intake (P<0·05). Multiple regression analysis showed that on most study days EE was more affected by energy intake than by weight gain. We conclude that in healthy preterm infants with birth weight <1000 g, EE increases by about 150 % in the first 5 weeks after birth, and that the EE values are related to energy intake and weight gain independent of postnatal age.

The objectives of the present work were to evaluate the effect of a methionine-supplemented diet as a model of hyperhomocysteinaemia on the systolic blood pressure (BP) and vasomotor functions of aortic rings in Wistar–Kyoto (WKY) and spontaneously hypertensive rats (SHR). WKY and SHR rats, randomised into four groups, were fed a normal semisynthetic diet or a methionine (8 g/kg)-supplemented diet for 10 weeks. Systolic BP was measured non-invasively. At the end of the experiment, plasma homocysteine, methionine, cysteine and glutathione levels were determined. Vasoconstriction and vasodilatation of aortic rings were measured. The methionine-supplemented diet induced a significant increase in plasma homocysteine and methionine concentration in both WKY and SHR rats, an increase in plasma cysteine concentrations in WKY rats and an increase in the glutathione concentration in SHR. The systolic BP of WKY rats fed the methionine-supplemented diet increased significantly (P<0·01), whereas systolic BP was reduced in SHR. An enhanced aortic responsiveness to noradrenaline and a decreased relaxation induced by acetylcholine and bradykinin were observed in the WKY rats fed the methionine-enriched diet. In SHR, the bradykinin-induced relaxation was reduced, but the sodium nitroprusside response was increased. In conclusion, a methionine-enriched diet induced a moderate hyperhomocysteinaemia and an elevated systolic BP in WKY rats that was consistent with the observed endothelial dysfunction. In SHR, discrepancies between the decreased systolic BP and the vascular alterations suggest more complex interactions of the methionine-enriched diet on the systolic BP. Further investigations are needed to understand the paradoxical effect of a methionine-rich diet on systolic BP.