Dietary carbohydrate rather than protein intake drives colonic microbial fermentation during weight loss.

PURPOSE: High protein weight loss diets are effective in aiding body weight management. However, high protein and low carbohydrate intakes can alter colonic fermentation profiles in humans and may impact on colonic health. This study aims to identify the most important dietary contributors to colonic fermentation during diet-controlled weight loss. METHODS: Overweight or obese male volunteers (n = 18) consumed a body weight maintenance diet (fed at 1.5× basic metabolic rate, BMR) followed by three weight loss diets (fed at 1× BMR) for 10 days each in a cross-over design. Weight loss diets were designed as normal protein (NPWL, 15% of energy from protein, 55% from carbohydrate), normal protein enriched with free amino acids and moderate amounts of carbohydrate (NPAAWL, 15% of energy from protein, 15% from free AA, 40% from carbohydrate) or high protein containing moderate amounts of carbohydrate (HPWL, 30% of energy from protein, 40% from carbohydrate). Faecal samples collected at the end of each diet period were profiled for dietary metabolites using LC-MS/MS. RESULTS: This study shows that the NPWL diet only induced very minor changes in the faecal metabolome, whereas NPAAWL and HPWL diets decreased carbohydrate-related metabolites (butyrate, ferulic acid) and increased protein-related metabolites. Most faecal metabolites were correlated with dietary carbohydrate and not protein intake. CONCLUSION: This study demonstrates that dietary carbohydrate is the main driver of colonic fermentation in humans and that a balance between dietary carbohydrate and protein should be maintained when designing safe, effective and healthy weight loss diets.

Whole-body and splanchnic amino acid metabolism in sheep during an acute endotoxin challenge.

Supplemented protein or specific amino acids (AA) are proposed to help animals combat infection and inflammation. The current study investigates whole-body and splanchnic tissue metabolism in response to a lipopolysaccharide (LPS) challenge with or without a supplement of six AA (cysteine, glutamine, methionine, proline, serine and threonine). Eight sheep were surgically prepared with vascular catheters across the gut and liver. On two occasions, four sheep were infused through the jugular vein for 20 h with either saline or LPS from Escherichia coli (2 ng/kg body weight per min) in a random order, plus saline infused into the mesenteric vein; the other four sheep were treated with saline or LPS plus saline or six AA infused via the jugular vein into the mesenteric vein. Whole-body AA irreversible loss rate (ILR) and tissue protein metabolism were monitored by infusion of [ring-2H2]phenylalanine. LPS increased (P<0·001) ILR (+17 %), total plasma protein synthesis (+14 %) and lymphocyte protein synthesis (+386 %) but decreased albumin synthesis (-53 %, P=0·001), with no effect of AA infusion. Absorption of dietary AA was not reduced by LPS, except for glutamine. LPS increased the hepatic removal of leucine, lysine, glutamine and proline. Absolute hepatic extraction of supplemented AA increased, but, except for glutamine, this was less than the amount infused. This increased net appearance across the splanchnic bed restored arterial concentrations of five AA to, or above, values for the saline-infused period. Infusion of key AA does not appear to alter the acute period of endotoxaemic response, but it may have benefits for the chronic or recovery phases.

Responses in whole-body amino acid kinetics to an acute, sub-clinical endotoxin challenge in lambs.

Some effects of parasitism, endotoxaemia or sepsis can be mitigated by provision of extra protein. Supplemented protein may encompass a metabolic requirement for specific amino acids (AA). The current study investigates a method to identify and quantify the amounts of AA required during inflammation induced by an endotoxin challenge. One of each pair of six twin sheep was infused in the jugular vein for 20 h with either saline (control) or lipopolysaccharide (LPS, 2 ng/kg body weight per min) from Escherichia coli. Between 12 and 20 h a mixture of stable isotope-labelled AA was infused to measure irreversible loss rates. From 16 to 20 h all sheep were supplemented with a mixture of unlabelled AA infused intravenously. Blood samples were taken before the start of infusions, and then continuously over intervals between 14 and 20 h. At 20 h the sheep were euthanised, and liver and kidney samples were taken for measurement of serine-threonine dehydratase (SDH) activity. LPS infusion decreased plasma concentrations of most AA (P<0·05; P<0·10 for leucine and tryptophan), except for phenylalanine (which increased P=0·022) and tyrosine. On the basis of the incremental response to the supplemental AA, arginine, aspartate, cysteine, glutamate, lysine (tendency only), glycine, methionine, proline, serine and threonine were important in the metabolic response to the endotoxaemia. The AA infusion between 16 and 20 h restored the plasma concentrations in the LPS-treated sheep for the majority of AA, except for glutamine, isoleucine, methionine, serine and valine. LPS treatment increased (P<0·02) SDH activity in both liver and kidney. The approach allows quantification of key AA required during challenge situations.

A randomized crossover study to assess the effect of an oat-rich diet on glycaemic control, plasma lipids and postprandial glycaemia, inflammation and oxidative stress in Type 2 diabetes.

AIMS: In the UK, lifestyle intervention is first-line management in Type 2 diabetes. It is unclear what type of diet is most efficacious for improving glycaemic control. This study investigated the effects of an oat-enriched diet on glycaemic control, postprandial glycaemia, inflammation and oxidative stress compared with standard dietary advice. METHODS: In a randomized crossover design, 27 volunteers with Type 2 diabetes, managed on diet and lifestyle only, were observed for two consecutive 8-week periods following either the oat-enriched diet or re-enforced standard dietary advice. Volunteers attended at baseline (habitual intake) and 8 and 16 weeks. Measurements included basic clinical measurements and fasted and postprandial (3-h) glucose and insulin in response to a healthy test meal. Markers of inflammation and oxidative stress, including high-sensitivity C-reactive protein, interleukin 6, interleukin 18, tumour necrosis factor-alpha, adiponectin, thiobarbituric acid reactive substances, oxygen radical antioxidant capacity, oxidized LDL and urinary isoprostanes, were also measured at fasting and in the postprandial period. RESULTS: There were no diet-related effects on glycaemic control or glycaemic or insulinaemic responses to the test meal. Total cholesterol (5.1 ± 1.0 vs. 4.9 ± 0.8 mmol/l, P = 0.019) concentrations declined following the oat-enriched diet compared with standard dietary advice. There was a postprandial decline in adiponectin concentration (P = 0.009), but no effect of dietary intervention. None of the measures of oxidative stress or inflammation were altered by the oat-enriched diet compared with standard dietary advice. CONCLUSION: The oat-enriched diet had a modest impact on lipid lowering, but did not impact on oxidative stress or inflammation in these volunteers with Type 2 diabetes.

Is D-methionine bioavailable to the dairy cow?

Rumen-protected forms of Met contain an equimolar mixture of the D- and L-isomers. Only L-Met can be directly used for protein synthesis, but it is unclear how much of the D-isomer can be transformed into L-Met in ruminants. Four lactating dairy cows, with an average milk yield of 32.4 kg/d, received a basal diet (12.5% crude protein, supplying 1,718 g/d of metabolizable protein) in 12 equal meals per day plus an abomasal infusion of amino acids (590 g/d, casein profile without Met). They were used in 3 consecutive studies to determine utilization of D-Met. First, the cows each received portal vein infusions for d of 5, 10, or 15 g/d of DL-Met in a Youden square. On the last day of each period, 6 arterial samples were collected at 45-min intervals. Concentrations of L- and D-Met were determined on a chiral column by gas chromatography-mass spectrometry. Portal infusion of 5, 10, and 15 g/d of DL-Met increased plasma total Met concentrations (19.7, 25.0, and 34.4±0.6 µM) and the proportion of Met as D (19.4, 30.5, and 37.3±0.7%). The fractional removal of D-Met was 6 to 7 times lower than the fractional removal of L-Met, with mean half-lives of 52 versus 8 min, respectively. Second, the same cows were infused for 8 h with L[methyl-(2)H(3)]Met at 1.3 mmol/h; at 2 h, cows received a bolus injection i.v. of D-[1-(13)C]Met (6.8 mmol), and arterial samples were collected after 10, 20, 30, 40, 60, 90, 120, 150, 180, 240, 300, 360, 420, and 480 min. Expressed relative to L-[(12)C]Met; that is, as tracer:tracee ratios, enrichments of plasma D-[1-(13)C]Met and L-[1-(13)C]Met averaged 1.77±0.14 and 0.144±0.026, respectively, 10 min after the bolus injection and declined exponentially thereafter. A minimum of 75±3% of the D-[1-(13)C]Met was transformed into L-[1-(13)C]Met. Third, the cows received, in a crossover design, an abomasal infusion for D of either DL-Met or L-Met (1g/d) and, on the last day of each experimental period, blood samples were collected simultaneously from arterial, portal, hepatic, and mammary vessels. Arterial total Met concentrations were higher with DL- versus L-Met infusions (37.4 vs. 25.4±0.5 µM), with 37.1±5.0% as D-Met. The mammary gland did not extract any D-Met. Hepatic removal of D-Met was observed, but was numerically lower than the fractional extraction of L-Met. In conclusion, much of the D-Met is transformed into L-Met by the dairy cow but at a slow rate. No uptake of D-Met occurs across the mammary gland but L-Met synthesized from the D-isomer elsewhere in the body can be utilized for milk protein synthesis.

Study of the effect of presence or absence of protozoa on rumen fermentation and microbial protein contribution to the chyme.

The aim of this study was to investigate the effect of presence or absence of protozoa on rumen fermentation and efficiency of microbial protein synthesis under different diets. Of 20 twin paired lambs, 1 lamb of each pair was isolated from the ewe within 24 h after birth and reared in a protozoa-free environment (n = 10), whereas their respective twin-siblings remained with the ewe (faunated, n = 10). When lambs reached 6 mo of age, 5 animals of each group were randomly allocated to 1 of 2 experimental diets consisting of either alfalfa hay as the sole diet, or 50:50 mixed with ground barley grain according to a 2 × 2 factorial arrangement of treatments. After 15 d of adaptation to the diet, the animals were euthanized and total rumen and abomasal contents were sampled to estimate rumen microbial synthesis using C(31) alkane as flow marker. Different ((15)N and purine bases) and a novel (recombinant DNA sequences) microbial markers, combined with several microbial reference extracts (rumen protozoa, liquid and solid associated bacteria) were evaluated. Absence of rumen protozoa modified the rumen fermentation pattern and decreased total tract OM and NDF digestibility in 2.0 and 5.1 percentage points, respectively. The effect of defaunation on microbial N flow was weak, however, and was dependent on the microbial marker and microbial reference extract considered. Faunated lambs fed with mixed diet showed the greatest rumen protozoal concentration and the least efficient microbial protein synthesis (29% less than the other treatments), whereas protozoa-free lambs fed with mixed diet presented the smallest ammonia concentration and 34% greater efficiency of N utilization than the other treatments. Although (15)N gave the most precise estimates of microbial synthesis, the use of recombinant DNA sequences represents an alternative that allows separate quantification of the bacteria and protozoa contributions. This marker showed that presence of protozoa decrease the bacterial-N flow through the abomasum by 33%, whereas the protozoa-N contribution to the microbial N flow increased from 1.9 to 14.1% when barley grain was added to the alfalfa hay. Absolute data related to intestinal flow must be treated with caution because the limitations of the sampling and maker system employed.

Metabolism of 2-hydroxy-4-(methylthio)butanoate (HMTBA) in lactating dairy cows.

The objectives of the current study were to determine the fate and contribution to Met kinetics of 2-hydroxy-4-(methylthio)butanoate (HMTBA) at the whole-body, splanchnic, and mammary levels. Four multicatheterized cows (31.3 kg of milk/d; 17.7 kg of DMI/d) were used in a crossover design, with two 1-wk periods, to determine the metabolic fate of HMTBA and its effect on Met metabolism. Over the last 2 d of each period, cows were infused, via a jugular vein, with saline or HMTBA (Alimet, Novus International Inc., St. Louis, Mo) at the rate of 36 g/d. During the last 8h, the HMTBA infusion was substituted by equimolar [1-(13)C]HMTBA (8.79 mmol/h) and l[methyl-(2)H(3)]Met (1.31 mmol/h) was infused in all cows. During the last 5h, hourly samples (n=6) were collected to determine plasma flows plus the isotopic enrichments (IE) and concentrations of HMTBA ((13)C) and Met (both (13)C and (2)H(3)) in plasma from an artery plus portal, hepatic, and mammary veins. The IE of [(13)C] and [(2)H(3)]Met were also determined in milk protein taken over the last 1h of infusion in HMTBA-infused cows. The infused HMTBA increased whole-body plasma flux of Met by 6.5 mmol/h (from 17.9 to 24.4 mmol/h). Based on enrichments of (13)C-labeled Met, 3.8 mmol/h of Met flow through plasma was derived directly from HMTBA. These 2 estimates accounted for between 43 to 74% of the HMTBA dose infused, contributing to increased whole-body Met availability. Although the portal-drained viscera, liver, and mammary gland (MG) extracted 11, 37, and 3.4%, respectively, of the infused HMTBA, tissue net Met fluxes were either unchanged (portal-drained viscera, MG) or even reduced (liver: -7.9 vs. -2.4±0.6 mmol/h). Therefore, net postsplanchnic supply of Met decreased from 7.0 to 2.9 mmol/h between control and HMTBA-infused cows, compared with needs for milk protein secretion of 7.6 and 8.1 mmol/h, respectively. The HMTBA provided directly 15% of the Met required for milk protein secretion, with 0.2 mmol/h synthesized within the MG, whereas 1.1 mmol/h originated from Met produced in other tissues and transported to the MG through blood circulation. Most of the remainder needed by the MG arose from unlabeled Met released from protein breakdown in extra-splanchnic tissues and that was not reused to support intracellular protein synthesis, as this function was performed by Met synthesized from HMTBA in situ. Absorbed HMTBA, therefore, both produces and spares Met for use by the MG.

Effect of method of conservation of timothy on endogenous nitrogen flows in lactating dairy cows.

The effect of the method of conservation of forage on endogenous N (EN) secretion was studied using a 15N isotope dilution technique in 4 lactating Holstein cows selected from a replicated 3x3 Latin square. Cows were equipped with ruminal, duodenal (n=4), and ileal (n=2) cannulas. Diets comprised 44% concentrate plus first-cut timothy conserved either as hay or as restrictively (formic) or extensively (inoc) fermented silage. Crude protein contents of hay, formic, and inoc averaged 10.4, 13.6, and 14.8%, respectively. Total EN flow and free EN at the duodenum were increased with hay compared with silages but were similar when expressed as proportion of duodenal N flow, with total EN flow averaging 25.8, 23.9, and 23.9% for hay, formic, and inoc, respectively, and free EN at the duodenum averaging 11.5, 9.8, and 9.7% for hay, formic, and inoc, respectively. Flow of bacterial N at the duodenum originating from an endogenous source tended to be higher with inoc compared with formic. Overall, the proportion of bacterial N derived from endogenous sources and urea was similar between treatments, averaging 23 and 15%, respectively. In the feces, flow of EN was similar across treatments and averaged 31% of total fecal N. More than 70% of fecal EN originated from undigested secretions into the forestomach. Absorption of N from the forestomach tended to increase for silages compared with hay. In conclusion, EN represented an important fraction of N flowing at the duodenum and in the feces. The free EN and the total EN at the duodenum were altered by the different methods of forage conservation studied. Estimation of true dietary N supply and requirements of the dairy cow should allow for endogenous N flows and losses.

Human colonic microbiota associated with diet, obesity and weight loss.

BACKGROUND: It has been proposed that the development of obesity in humans is influenced by the relative proportions of the two major phyla of bacteria (Bacteroidetes and Firmicutes) present in the large intestine. OBJECTIVE: To examine the relationships between body mass index, weight loss and the major bacterial groups detected in fecal samples. DESIGN: Major groups of fecal bacteria were monitored using fluorescent in situ hybridization (FISH) in obese and non-obese subjects under conditions of weight maintenance, and in obese male volunteers undergoing weight loss on two different reduced carbohydrate weight-loss diets given successively for 4 weeks each. RESULTS: We detected no difference between obese and non-obese individuals in the proportion of Bacteroidetes measured in fecal samples, and no significant change in the percentage of Bacteroidetes in feces from obese subjects on weight loss diets. Significant diet-dependent reductions in a group of butyrate-producing Firmicutes were, however, detected in fecal samples from obese subjects on weight loss diets. CONCLUSIONS: Diets designed to achieve weight loss in obese subjects can significantly alter the species composition of the gut microbiota, but we find no evidence that the proportions of Bacteroidetes and Firmicutes among fecal bacteria have a function in human obesity.

Distribution of 15N in amino acids during 15N-leucine infusion: impact on the estimation of endogenous flows in dairy cows.

The distribution of (15)N in AA during [(15)N]Leu infusion and its impact on the estimation of endogenous nitrogen (EN) flows in dairy cows was evaluated in 4 lactating cows equipped with ruminal, duodenal (n = 4), and ileal (n = 2) cannulae fed a silage-based diet during a 35-d experimental period. To label EN, starting on d 27, an infusion of L-[(15)N]Leu (0.45 mmol/h) was performed for 200 h. Samples of feed, duodenal and ileal digesta, feces, blood, urine, and mucosa of the rumen and duodenum were taken at 0900, 1100, 1300, and 1500 h on d 34 and at 0800, 1000, 1200, and 1400 h on d 35. The enrichment and fluxes of total N and individual AA were determined and used to calculate the EN flows at the duodenum, ileum, and in the feces. Based on the concept that EN comprises desquamation and secretions, EN flows were estimated, using as representative of the enrichment of EN only the enrichment of the gut mucosa (upper limit) or the average of the mucosa and the export protein enrichment (assumed to have a similar enrichment to casein; lower limit). Estimations of duodenal and fecal EN flows using the isotope dilution of (15)N-total and (15)N-Leu were not different and EN was an important fraction of duodenal and fecal flows, representing 14 to 30% of the duodenal flow and 18 to 31% of the fecal flow, depending on the dilution method used. The total EN flow at the duodenum is present in approximately equal proportions as either free EN or EN incorporated into bacterial protein. Ileal EN flow was 18% greater than the fecal EN flow. Using the combination of the gut and export protein, the duodenal and fecal EN flows estimated with the isotopic dilution of Leu vs. other labeled AA were less different than when estimated using the enrichment of gut mucosa alone. The current approaches have highlighted that present prediction schemes probably underestimate EN flows at the duodenum and, in consequence, overestimate net protein and AA supply. Refinement of the procedures may allow direct and accurate estimation of metabolic fecal protein, an important component of the so-called maintenance requirement of dairy cows.

Absorption and digestive tract metabolism of 2-hydroxy-4-methylthiobutanoic acid in lambs.

Anabolic availability of the hydroxyl methionine analog, 2-hydroxy-4-methylthiobutanoic acid (HMTBA), given as oral doses to lambs, was quantified both directly as appearance in the portal vein and as synthesis to Met by digestive tract tissues. Eight lambs, prepared with vascular catheters in the mesenteric and portal veins plus the aorta, received twice daily for 7 d either 0.46 g or 2 g of HMTBA. On d 7, [1-13C]HMTBA was supplied as 1 oral dose while [methyl-2H3]Met was infused into the jugular vein. Peak absorption as HMTBA occurred 70 to 90 min after the oral dose. All digestive tract tissues converted HMTBA to Met, equivalent to 24% of the Met provided by the diet for the larger HMTBA dose. Overall, total availability of HMBTA averaged 17.9% of the dose (range 10.6 to 27.9%), with 12.5% (range 7 to 22%) as absorbed HMBTA and the remainder as Met synthesized by digestive tract tissues. Release of 13CO2 into the portal vein accounted for another 23% of the dose. In all digestive tract tissues, the d-isomer was present in a smaller proportion than in the dose. In terms of whole-body kinetics, HMTBA loss from the plasma followed first-order kinetics, with a mean biological half-life of 76 min. Using this value, a simple model was devised to estimate HMTBA absorption based on peripheral plasma samples. When compared with direct measures of absorption, the model gave a slope of 0.81 (R2 = 0.68) and offers a practical means to test HMTBA availability to animals.

What is the true supply of amino acids for a dairy cow?

Improving the prediction of milk protein yield relies on knowledge of both protein supply and requirement. Definition of protein/amino acid supply in ruminants is a challenging task, due to feedstuff variety and variability and to the remodeling of nutrient intake by the rumen microflora. The questions arise, therefore, how and where should we measure the real supply of AA in the dairy cow? This review will follow the downstream flow of AA from duodenum to peripheral tissue delivery, with a glance at the efficiency of transfer into milk protein. Duodenal AA flow comprises rumen undegradable feed, microbial protein, and endogenous secretions. Most attention has been directed toward definition of the first two contributions but the latter fraction can represent as much as 20% of duodenal flow. More information is needed on what factors affect its magnitude and overall impact. Once digested, AA are absorbed into the portal vein. The ratio of portal absorption to small intestinal apparent digestion varies among essential AA, from 0.43 (threonine) to 0.76 (phenylalanine), due to the contributions of preduodenal endogenous secretions to the digestive flow, non-reabsorption of endogenous secretions and gut oxidation of AA. Few data are available on these phenomena in dairy cows but the evidence indicates that they alter the profile of AA available for anabolic purposes. Recent comparisons of estimated duodenal flux and measured portal flux have prompted a revisit of the NRC (2001) approach to estimate AA flows at the duodenum. Changes to the model are proposed that yield predictions that better fit the current knowledge of AA metabolism across the gut. After absorption, AA flow first to the liver where substantial and differential net removal occurs, varying from zero for the branched-chain AA to 50% of portal absorption for phenylalanine. This process alters the pattern of net supply to the mammary gland. Overall, intermediary metabolism of AA between the duodenum and the mammary gland biologically explains the decreased efficiency of the transfer of absorbed AA into milk protein as maximal yield is approached. Therefore, variable, rather than fixed, factors for transfer efficiencies must be incorporated into future predictive models.

Can general anaesthesia be used for the Paralytic Shellfish Poison bioassay?

The current method for Paralytic Shellfish Poisoning (PSP) testing in shellfish is based on the mouse bioassay (MBA), which involves injecting shellfish extract into a conscious mouse, and then converting its time to death into PSP toxicity using Sommer's table. To improve animal welfare, the present study investigated the use of anaesthesia. A saxitoxin (STX) calibration study was conducted where known amounts of STX were injected into both unanaesthetised and anaesthetised mice. Death time was approximately doubled when mice were anaesthetised. Both unanaesthetised and anaesthetised animals showed a linear relationship between the inverse death time and log(STX). Based on these data, new calibration curves were developed. This study revealed that the current method employing Sommer's table underestimates toxicity by up to 50% for higher toxin levels. Subsequently, shellfish samples were tested on both unanaesthetised and anaesthetised mice. Using the new calibration curves, the numbers of samples exceeding the field closure limit were similar for unanaesthetised and anaesthetised mice, and were nearly two-fold higher than those obtained with the current method. The studies showed that the bioassay gives variable results for both unanaesthetised and anaesthetised animals. Anaesthesia forms a viable and more ethical alternative to the current bioassay, at least in the short term. A practical summary on how to conduct this method is given.

[The transmandibular implant. From bone loss to bone growth--an investigation of 116 patients]. / Het transmandibulaire implantaat. Van botverlies tot botgroei--een onderzoek bij 116 patiënten.

In a prospective study 116 consecutive patients were treated according to a revised protocol to induce and control bone growth. Revisions included: 1. lengthening of the transosseous posts and cortical screws between the mental foramina so that two threads extended beyond the alveolar crest of the mandible without protruding through the mucosa; and 2. fabricating an implant borne prosthesis with a gap of 2 mm between the denture base and the mucosal tissues in the saddle areas and loading only the retromolar pads. The gap was re-opened every 8 weeks until further bone growth would prevent proper oral hygiene. Measurements of the height of the mandible were made using a digital millimeter caliper and standardized radiographs. The radiographic enlargement was calculated per radiograph for the sites to be measured. The follow-up varied from 15 up to 39 months. Bone growth had occurred in 104 of the 116 patients, while the resorption of bone had ceased in the remaining patients. The increase of bone height varied from 9 mm in patients with severe mandibular atrophy down to 2 mm for patients with mild atrophy. The revised protocol for TMI insertion and rehabilitation is advocated to promote bone growth and to cease further resorption in the atrophic mandible.