1. Measurements were made, at 6 h intervals, of urinary nitrogen output and of the activity of some hepatic enzymes in the rat during adaptation from one level of dietary protein to another. The enzymes measured were arginase (EC 3.5.3.1), argininosuccinate lyase (EC 4.3.2.1), argininosuccinate synthetase (EC 6.3.4.5), glutamate dehydrogenase (EC 1.4.1.2) and alanine and aspartate aminotransferases (EC 2.6.1.2 and EC 2.6.1.1).

2. Completeness of urine collection, which was essential for these experiments, was checked by recovery of injected [131I]iodide.

3. When the dietary protein content was reduced from 135 to 45 g casein/kg, the urinary N output and the activities of the hepatic enzymes reached their new steady-state levels in 30 h. The reverse adaptation, from 45 to 135 g casein/kg, was also complete in 30 h.

4. The rate of change of enzyme activity and the final activity as percentage of initial activity were very similar for all six enzymes, suggesting a common control mechanism. The calculated half-lives of the enzymes were of the order of 7 h, which is very much shorter than those found by previous workers.

5. There was no simple relationship between the activity of the urea cycle enzymes and the amount of N excreted. When an equal amount of gelatin was substituted for casein the N output was doubled but there was no change in the activity of the liver enzymes.

6. The results suggest that the activity of the urea cycle enzymes depends in part on the amount of N available for excretion after the demands for synthesis have been met. The enzymes, however, appear to be present in excess so that an increased N load was not necessarily accompanied by an increase in enzyme activity.

Background & Aim: Despite their well known anti-inflammatory actions, the clinical usefulness of omega-3 PUFA in inflammatory bowel disease is controversial. We aimed to systematically review the available data on the performance of omega-3 PUFA as therapeutic agents in these patients. Methods: Electronic databases were systematically searched for RCT of fish oil or omega-3 PUFA therapy in both active and inactive ulcerative colitis or Crohn's disease, without limitation on either the length of therapy or the form it was given, including nutritional supplements and enteral formula diets. Eligible articles were assessed for methodological quality on the basis of the adequacy of the randomisation process, concealment of allocation, blinding of intervention and outcome, possible biases, and completeness of follow-up. The five-point Oxford quality score was calculated. Results: A total of 19 RCT were finally selected for this review. Overall, available data do not allow to support the use of omega-3 PUFA supplementation for the treatment of both active and inactive inflammatory bowel disease. Negative results are quite consistent in trials assessing the use of omega-3 PUFA to maintain disease remission, particularly ulcerative colitis, and to a lesser extent Crohn's disease. Trials on their use in active disease do not allow to draw firm conclusions mainly because the heterogeneity of design (ulcerative colitis) or their short number (Crohn's disease). In most trials, the appropriateness of the selected placebo is questionable. Conclusion: The present systematic review does not allow to make firm recommendations about the usefulness of omega-3 PUFA in inflammatory bowel disease.

Overnutrition, insulin resistance and an impaired intestinal barrier function are discussed as critical factors in the development of non-alcoholic fatty liver disease. Not only butyrate-producing probiotics as well as supplementation of sodium butyrate (SoB) have been suggested to bear protective effects on liver damage of various aetiologies. However, whether an oral consumption of SoB has a protective effect on Western-style diet (WSD)-induced non-alcoholic steatohepatitis (NASH) and if so molecular mechanism involved has not yet been determined. Eight-week-old C57BL/6J mice were pair-fed either a liquid control or WSD±0·6 g/kg body weight SoB. After 6 weeks, markers of liver damage, inflammation, toll-like receptor (TLR)-4 signalling, lipid peroxidation and glucose as well as lipid metabolism were determined in the liver tissue. Tight junction protein levels were determined in the duodenal tissue. SoB supplementation had no effects on the body weight gain or liver weight of WSD-fed mice, whereas liver steatosis and hepatic inflammation were significantly decreased (e.g. less inflammatory foci and neutrophils) when compared with mice fed only a WSD. Tight junction protein levels in duodenum, hepatic mRNA expression of TLR-4 and sterol regulatory element-binding protein 1c were altered similarly in both WSD groups when compared with controls, whereas protein levels of myeloid differentiation primary response gene 88, inducible nitric oxide synthase, 4-hydroxynonenal protein adducts and F4/80 macrophages were only significantly induced in livers of mice fed only the WSD. In summary, these data suggest that an oral supplementation of SoB protects mice from inflammation in the liver and thus from the development of WSD-induced NASH.

Fractured neck of femur occurs mostly in the older female population and is generally caused by falls. Malnutrition has been postulated as a factor that increases the tendency to suffer falls. Nutritional status of older female hospital patients admitted for emergency surgery for fractured neck of femur recruited (n 75), was compared with an age-matched independent-living group of females attending one of three local day centres (n 50). Dietary assessment was undertaken using three consecutive 24 h dietary recalls and, in the hip fracture group, completed menu cards were used as memory prompts. Data concerning key lifestyle characteristics were obtained using a face-to-face administered questionnaire. Blood samples were taken to determine levels of plasma albumin, transferrin, C-reactive protein (CRP), cholesterol, vitamin C, Se, Zn and total antioxidant status. Haemolysate samples were analysed for Se-dependent glutathione peroxidase activity. There were no significant differences in age between the two groups, but the hip fracture patients had lower mean values for body weight (59.6 v. 67.5 kg; p = 0.005), mindex (weight/demispan) (83· v. 94.4 kg/m; P < 0·0001), calculated BMI (24·1 v. 27·5 kg/m2P<0·0001), mid-upper arm circumference; 27·1 v. 31·3 cm, p = 0.001) and triceps skinfold thickness; 17·0 v. 18.9 mm, p = 0·005) than the home-living group. The hip fracture patients had lower intakes of energy (4·3 v. 5·4 MJ, p = 0·001), fat (p = 0·025), carbohydrate (p = 0·002), protein (p = 0·006), thiamine (p = 0·017), vitamin B6 (p = 0·001), calcium (p = 0·01), K (p = 0·001), Mg (p = 0·001), P (p = 0·001), Fe (p = 0·007), Se (p = 0·008) and NSP (p = 0·001). Mean intakes of both groups were below the estimated average requirement for energy and below the reference nutrient intakes for folate, Ca, vitamin D, Mg, K, Se and Zn. In a high percentage of the hip fracture group the dietary intake of particular nutrients fell below the lower reference nutrient intake for Se (73 %), Mg (54 %) and Fe (19 %). As expected, the fracture patients had reduced plasma albumin (P<0·0001) and increased CRP (P<0·001) values. They had higher plasma vitamin C levels (P<0·001) and lower cholesterol levels (p = 0.04) than the day centre attendees. There were no significant differences in plasma levels of Se, Zn, transferrin or haemolysate glutathione peroxidase activity between the two groups. However, there was evidence of under-nutrition in both groups as key anthropometric values were low, plasma nutrient and metabolite levels were below the standard reference ranges and many individuals had low dietary intakes for specified nutrients.

Dietary cholesterol comes exclusively from animal sources, thus it is naturally present in our diet and tissues. It is an important component of cell membranes and a precursor of bile acids, steroid hormones and vitamin D. Contrary to phytosterols (originated from plants), cholesterol is synthesised in the human body in order to maintain a stable pool when dietary intake is low. Given the necessity for cholesterol, very effective intestinal uptake mechanisms and enterohepatic bile acid and cholesterol reabsorption cycles exist; conversely, phytosterols are poorly absorbed and, indeed, rapidly excreted. Dietary cholesterol content does not significantly influence plasma cholesterol values, which are regulated by different genetic and nutritional factors that influence cholesterol absorption or synthesis. Some subjects are hyper-absorbers and others are hyper-responders, which implies new therapeutic issues. Epidemiological data do not support a link between dietary cholesterol and CVD. Recent biological data concerning the effect of dietary cholesterol on LDL receptor-related protein may explain the complexity of the effect of cholesterol on CVD risk.

Three xylanase products, xylanase A derived from Thermomyces lanuginosus, xylanase B from Humicola insolens and xylanase C from Aspergillus aculeatus, were examined for their effects on the nutritive value of wheat. The study investigated the effects of enzyme addition to broiler diets based on a low-metabolisable-energy (ME) wheat and a normal-ME wheat, with the emphasis on changes in composition of the NSP along the digestive tract in broiler chickens. There were significant (P>0·01) enzyme and wheat effects on the apparent ME, but there was no wheat×enzyme interaction on apparent ME. Weight gain and feed conversion efficiency (except xylanase C for normal wheat) were also significantly (P>0·01) increased by the xylanases. The enzymes differed in their effect on a number of important nutritional parameters. Xylanase A reduced (P>0·05) the excreta moisture level from 77·1% in birds fed the normal-ME wheat control to 73·4% and from 77·4% in those fed the low-ME wheat to 73·0%. The other two enzymes did not affect excreta moisture levels. The digesta viscosity in the duodenum, jejunum and ileum of birds fed both types of wheat was reduced (P>0·01) by xylanases A and C, but was increased (P>0·01) in the jejunum and ileum by xylanase B. The digestibility of the soluble NSP was mostly negative in the small intestine. The digestibility of the insoluble NSP differed (P>0·01) between the two wheats, with those in the low-ME wheat being more digestible (14 v. 28%). When supplemented with xylanase A, the ileal digestibility of the insoluble NSP in the normal- and low-ME wheats was increased by 28 and 42?%, respectively. Xylanases B and C did not affect the digestibility of the insoluble NSP regardless of wheat type. It may be concluded that wheats with low or normal ME values vary in their responses to xylanase supplementation. Apart from having an elevated level of soluble NSP, low-ME wheat may also contain insoluble NSP, which in the present study appeared to be more easily degradable in the gut of the chicken.

Recently, novel dietary oils with modified fatty acid profiles have been manufactured to improve fatty acid intakes and reduce CVD risk. Our objective was to evaluate the efficacy of novel high-oleic rapeseed (canola) oil (HOCO), alone or blended with flaxseed oil (FXCO), on circulating lipids and inflammatory biomarkers v. a typical Western diet (WD). Using a randomised, controlled, crossover trial, thirty-six hypercholesterolaemic subjects consumed three isoenergetic diets for 28 d each containing approximately 36 % energy from fat, of which 70 % was provided by HOCO, FXCO or WD. Dietary fat content of SFA, MUFA, PUFA n-6 and n-3 was 6, 23, 5, 1 % energy for HOCO; 6, 16, 5, 7·5 % energy for FXCO; 11·5, 16, 6, 0·5 % energy for WD. After 28 d, compared with WD, LDL-cholesterol was reduced 15·1 % (P < 0·001) with FXCO and 7·4 % (P < 0·001) with HOCO. Total cholesterol (TC) was reduced 11 % (P < 0·001) with FXCO and 3·5 % (P = 0·002) with HOCO compared with WD. Endpoint TC differed between FXCO and HOCO (P < 0·05). FXCO consumption reduced HDL-cholesterol by 8·5 % (P < 0·001) and LDL:HDL ratio by 7·5 % (P = 0·008) v. WD. FXCO significantly decreased E-selectin concentration compared with WD (P = 0·02). No differences were observed in inflammatory markers after the consumption of HOCO compared with WD. In conclusion, consumption of novel HOCO alone or when blended with flaxseed oil is cardioprotective through lipid-lowering effects. The incorporation of flaxseed oil may also target inflammation by reducing plasma E-selectin.

Inulin, a linear β fructan, is present in a variety of plants including chicory root and wheat. It exhibits prebiotic properties and has been shown to enhance mineral absorption and increase beneficial bacteria in the colon. The aim of the present study was to assess the effect of dietary inulin on the gene expression of selected intestinal Fe transporters and binding proteins. Anaemic piglets at age 5 weeks were allocated to a standard maize–soya diet (control) or the same diet supplemented with inulin at a level of 4 %. After 6 weeks, the animals were killed and caecum contents and sections of the duodenum and colon were removed. Segments of the genes encoding for the pig divalent metal transporter 1 (DMT1) and duodenal cytochrome-b reductase (Dcytb) were isolated and sequenced. Semi-quantitative RT-PCR analyses were performed to evaluate the expression of DMT1, Dcytb, ferroportin, ferritin, transferrin receptor (TfR) and mucin genes. DMT1, Dcytb, ferroportin, ferritin and TfR mRNA levels in duodenal samples were significantly higher in the inulin group (P ≤ 0·05) compared with the control. In colon, DMT1, TfR and ferritin mRNA levels significantly increased in the inulin group. Additionally, the caecal content microflora was examined using 16S rDNA targeted probes from bacterial DNA. The Lactobacillus and Bifidobacterium populations were significantly increased in the inulin group (P ≤ 0·05) compared with the control group. These results indicate that dietary inulin might trigger an up regulation of genes encoding for Fe transporters in the enterocyte. The specific mechanism for this effect remains to be elucidated.

To determine the optimum indispensable (I) amino acid (AA) balance in Atlantic salmon (Salmo salar L.) fry, a single protocol established for the pig was adapted. The balance was calculated from the reduction in N gain after replacing about 45% of a single IAA by a mixture of dispensable AA in isonitrogenous diets. We confirmed that the mixture of AA simulating the AA pattern of cod-meal protein and gelatine (46:3, w/w) was used with the same efficiency as cod-meal protein and gelatine. From the deletion experiment an optimum balance between the IAA was derived. Expressed relative to lysine=100, the optimal balance was: arginine 76 (se 0·2), histidine 28 (se 2·2), methionine+cystine 64 (se 1·7), phenylalanine + tyrosine 105 (se 1·6), threonine 51 (se 2·4), tryptophan 14 (se 0·7), valine 59 (se 1·7). No estimates were made for isoleucine and leucine. Expressed as g/16g N, the optimal balance was: arginine 4·0 (se 0·0), histidine 1·5 (se 0·1), lysine 5·3 (se 0·2), methionine+cystine 3·4 (se 0·1), phenylaline+tyrosine 5·6 (se 0·1), threonine 2·7 (se 0·1), tryptophan 0·7 (se 0·0), valine 3·1 (se 0·1). This AA composition is close to that of the Atlantic salmon whole-body, but using it as an estimation of the IAA requirements may lead to an overestimation of the branched-chain AA requirements and an underestimation of aromatic and S-containing AA requirements. The results are discussed in accordance with the key assumptions associated with the model used (broken-line model, IAA efficiencies and maintenance requirements).

Chokeberries are a rich source of anthocyanins, which may contribute to the prevention of obesity and the metabolic syndrome. The aim of the present study was to determine if an extract from chokeberries would reduce weight gain in rats fed a fructose-rich diet (FRD) and to explore the potential mechanisms related to insulin signalling, adipogenesis and inflammatory-related pathways. Wistar rats were fed a FRD for 6 weeks to induce insulin resistance, with or without chokeberry extract (CBE) added to the drinking-water (100 and 200 mg/kg body weight, daily: CBE100 and CBE200). Both doses of CBE consumption lowered epididymal fat, blood glucose, TAG, cholesterol and LDL-cholesterol. CBE consumption also elevated plasma adiponectin levels and inhibited plasma TNF-α and IL6, compared with the control group. There were increases in the mRNA expression for Irs1, Irs2, Pi3k, Glut1, Glut4 and Gys1, and decreases in mRNA levels of Gsk3β. The protein and gene expression of adiponectin and Pparγ mRNA levels were up-regulated and Fabp4, Fas and Lpl mRNA levels were inhibited. The levels of gene expression of inflammatory cytokines, such as Il1β, Il6 and Tnfα were lowered, and protein and gene expression of ZFP36 (zinc finger protein) were enhanced in the epididymal adipose tissue of the rats that consumed the CBE200 extract. In summary, these results suggest that the CBE decreased risk factors related to insulin resistance by modulating multiple pathways associated with insulin signalling, adipogenesis and inflammation.

Increasingly, the microbiological scientific community is relying on molecular biology to define the complexity of the gut flora and to distinguish one organism from the next. This is particularly pertinent in the field of probiotics, and probiotic therapy, where identifying probiotics from the commensal flora is often warranted. Current techniques, including genetic fingerprinting, gene sequencing, oligonucleotide probes and specific primer selection, discriminate closely related bacteria with varying degrees of success. Additional molecular methods being employed to determine the constituents of complex microbiota in this area of research are community analysis, denaturing gradient gel electrophoresis (DGGE)/temperature gradient gel electrophoresis (TGGE), fluorescent in situ hybridisation (FISH) and probe grids. Certain approaches enable specific aetiological agents to be monitored, whereas others allow the effects of dietary intervention on bacterial populations to be studied. Other approaches demonstrate diversity, but may not always enable quantification of the population. At the heart of current molecular methods is sequence information gathered from culturable organisms. However, the diversity and novelty identified when applying these methods to the gut microflora demonstrates how little is known about this ecosystem. Of greater concern is the inherent bias associated with some molecular methods. As we understand more of the complexity and dynamics of this diverse microbiota we will be in a position to develop more robust molecular-based technologies to examine it. In addition to identification of the microbiota and discrimination of probiotic strains from commensal organisms, the future of molecular biology in the field of probiotics and the gut flora will, no doubt, stretch to investigations of functionality and activity of the microflora, and/or specific fractions. The quest will be to demonstrate the roles of probiotic strains in vivo and not simply their presence or absence.

Previous studies have suggested that probiotic fermented milk may possess blood pressure (BP)-lowering properties. In the present study, we aimed to systematically examine the effect of probiotic fermented milk on BP by conducting a meta-analysis of randomised controlled trials. PubMed, Cochrane library and the ClinicalTrials.gov databases were searched up to March 2012 to identify eligible studies. The reference lists of the obtained articles were also reviewed. Either a fixed-effects or a random-effects model was used to calculate the combined treatment effect. Meta-analysis of fourteen randomised placebo-controlled trials involving 702 participants showed that probiotic fermented milk, compared with placebo, produced a significant reduction of 3·10 mmHg (95 % CI − 4·64, − 1·56) in systolic BP and 1·09 mmHg (95 % CI − 2·11, − 0·06) in diastolic BP. Subgroup analyses suggested a slightly greater effect on systolic BP in hypertensive participants than in normotensive ones ( − 3·98 v. − 2·09 mmHg). Analysis of trials conducted in Japan showed a greater reduction than those conducted in European countries for both systolic BP ( − 6·12 v. − 2·08 mmHg) and diastolic BP ( − 3·45 v. − 0·52 mmHg). Some evidence of publication bias was present, but sensitivity analysis excluding small trials that reported extreme results only affected the pooled effect size minimally. In summary, the present meta-analysis suggested that probiotic fermented milk has BP-lowering effects in pre-hypertensive and hypertensive subjects.

Six dairy cows of two breeds were fed during three alternating periods with products from C3- and C4-plants to yield different natural 13C enrichments of the diet (δ13C range: -28.0 to -13.7‰). The resulting changes in the 13C enrichment of breath carbon dioxide, serum and milk of the animals followed the 13C: 12C of the food, in agreement with the individual biological half-lives of those products, and established isotope discriminations. Breath CO2 was more enriched in 13C than expected. This could be related to isotope discriminations during rumen fermentation. From these results an isotopic balance model for the breath CO2 could be established.

Colorectal cancer is one of the most important causes of cancer morbidity and mortality in Western countries. While a myriad of healthful effects have been attributed to the probiotic lactic acid bacteria (LAB), perhaps the most controversial remains that of anticancer activity. It should be pointed out that there is no direct experimental evidence for cancer suppression in man as a result of consumption of lactic cultures in fermented or unfermented dairy products. However, there is a wealth of indirect evidence, based largely on laboratory studies, in the literature. The precise mechanisms by which LAB may inhibit colon cancer are presently unknown. However, such mechanisms might include: alteration of the metabolic activities of intestinal microflora; alteration of physico-chemical conditions in the colon; binding and degrading potential carcinogens; quantitative and/or qualitative alterations in the intestinal microflora incriminated in producing putative carcinogen(s) and promoters (e.g. bile acid-metabolising bacteria); production of antitumourigenic or antimutagenic compounds; enhancing the host's immune response; and effects on physiology of the host. These potential mechanisms are addressed in the present paper.

The beneficial influences of xylo-oligosaccharides (XOS) obtained from alkali-pretreated corncob and fructo-oligosaccharides (FOS) obtained from cane sugar were evaluated in experimental diabetes. These oligosaccharides were supplemented at 10 % (w/w) in the basal diet of streptozotocin-induced diabetic Wistar rats, while the control rats were fed with a basal diet for a period of 6 weeks. Both the oligosaccharides exerted favourable influences in diabetic rats by significantly improving body weight and reducing hyperglycaemia and cholesterol. The characteristic diabetic complications such as severe glucosuria, proteinuria and advanced glycation end products in renal tissue, diabetic nephropathy, and blood creatinine and urea concentrations were notably reduced. Besides, these oligosaccharide supplementations significantly increased the activity of antioxidant enzymes – catalase and glutathione reductase – in the blood of diabetic rats. Supplementation of XOS and FOS resulted in a significant increase in the bifidobacteria and lactobacilli population in the caecum. The present study indicates that XOS and FOS have an ameliorating influence on metabolic abnormalities associated with diabetes, besides conferring an optimal milieu of lactobacilli and bifidobacteria, thus suggesting their potential health benefit in diabetics.

In order to investigate the effects of dietary fibre (DF) characteristics on carbohydrate degradation and the metabolism in the large intestine, pigs were fed on four rye-bread diets (based on whole rye, pericarp/testa, aleurone or endosperm) with differences in characteristics and amount of DF. The degradability of DF in the large intestine varied greatly between diets. The pericarp/testa DF was hardly degraded in the large intestine, whereas endosperm DF was extensively and rapidly degraded in the caecum. Caecal degradation of aleurone DF was also limited, leaving more material to be degraded in the colon. The undegradable pericarp/testa DF was characterized by high contents of lignin, cellulose, ferulic acids and highly substituted arabinoxylans (the major DF component in rye). Ingestion of this diet resulted in increased faecal bulk and reduced transit time, but with low colonic pH and the lowest concentrations of short-chain fatty acids (SCFA). The aleurone diet, on the other hand, led to a fermentation pattern which may be considered more optimal, with lower colonic pH and higher concentrations of SCFA, in particular butyric acid. Despite the large differences in carbohydrate fermentation only minor significant effects on the presence of protein degradation products and on histological measurements (height and diameter of colonic crypts and thickness of the muscularis externa) were observed.

This study was conducted to elucidate the effects of decaffeinated green coffee bean extract (GCE) on anthropometric indices, glycaemic control, blood pressure, lipid profile, insulin resistance and appetite in patients with the metabolic syndrome (Mets). Subjects were randomly allocated to consume 400 mg GCE or placebo capsules twice per d for 8 weeks. Both groups were advised to follow an energy balanced diet. After GCE supplementation, systolic blood pressure (SBP) significantly reduced compared with the placebo group (−13·76 (sd 8·48) v. −6·56 (sd 9·58) mmHg, P=0·01). Also, GCE treatment significantly reduced fasting blood glucose (FBS) (−5·15 (sd 60·22) v. 29·42 (sd 40·01) mg/dl (−0·28 (SD 3·34) v. 1·63 (SD 2·22) mmol/l); P=0·03) and homoeostatic model of assessment of insulin resistance in comparison to placebo (−1·41 (sd 3·33) v. 1·23 (sd 3·84), P=0·02). In addition, waist circumference (−2·40 (sd 2·54) v. −0·66 (sd 1·17) cm, P=0·009) and appetite score (−1·44 (sd 1·72) v. −0·2 (sd 1·32), P=0·01) of the individuals supplemented with GCE indicated a significant decline. Besides, weight and BMI reduction in the intervention group was almost twice as much as the placebo group; however, this discrepancy was marginally significant (weight: −2·08 (sd 2·11) v. −0·92 (sd 1·30) kg, P=0·05). No difference was observed in terms of glycated Hb (HbA1c) percentage and lipid profile parameters between the two groups. To sum up, GCE administration had an ameliorating effect on some of the Mets components such as high SBP, high FBS and Mets main aetiological factors including insulin resistance and abdominal obesity. Furthermore, GCE supplementation could reduce appetite level.

Milk fat is high in saturated fatty acids (SFA) and high intakes of SFA are associated with cardiovascular diseases. The aim of the present study was to prospectively evaluate the potential risk of a first-ever acute myocardial infarction (AMI) in relation to the estimated milk-fat intake, reflected as the proportions of pentadecanoic acid (15:0) and heptadecanoic acid (17:0) in serum lipid esters. This was evaluated in a study population selected within the Västerbotten Intervention Program and the northern Sweden ‘Monitoring of Trends and Determinants in Cardiovascular disease’ survey populations. A prospective case–control design was used. The proportions of the biomarkers were lower in the cases (n78) than in the controls (n156), who were matched for age, sex, sampling time and geographical region. The standardised odds ratios of becoming an AMI case were between 0·7 and 0·8 for the biomarkers. The proportions of 15:0 and 17:0 in serum phospholipids were significantly and negatively correlated to serum concentrations of plasminogen activator inhibitor-1, tissue-type plasminogen activator, triacylglycerols, insulin, specific insulin, pro-insulin and leptin (all P<0·0001), suggesting a negative relationship to the insulin-resistance syndrome and the risk of CHD. Adjustment for BMI did not materially change the relationships. Although there seems to be a negative association between milk-fat intake as mirrored by the proportions of 15:0 and 17:0 in serum lipid esters and a first-ever AMI, adjustment for clinical risk factors removed this relationship.

1. A mathematical treatment is given of the marker technique for studying water balances in the rumen. The treatment is extended to cover non-steady-state conditions and emphasis is placed on the underlying assumptions and practical limitations of the technique.

2. The 51Cr complex of ethylenediamine tetra-acetic acid (51Cr EDTA) was used as a marker in the rumen of sheep.

3. The 51Cr EDTA entered 95–98% of the water in the rumen. Mixing in the rumen, though adequate, was usually not quite complete even 1·5 h after injection of the dose. The techniques of injecting the dose and of withdrawing the sample were found to be important; some techniques often led to poor mixing.

4. Provided that the time of injection of the dose was suitably selected, reliable estimates of the volume of water in the rumen could readily be made, but reliable estimates of flow rates needed stringent attention to details of technique.