Effects of non-digestible carbohydrates on gut microbiota and microbial metabolites: a randomised, controlled dietary intervention in healthy individuals.

The gut microbiome is impacted by certain types of dietary fibre. However, the type, duration, and dose needed to elicit gut microbial changes, and whether these changes also influence microbial metabolites, remains unclear. This study investigated the effects of supplementing healthy participants with two types of non-digestible carbohydrates (resistant starch (RS) and polydextrose (PD)), on the stool microbiota and microbial metabolite concentrations in plasma, stool, and urine, as secondary outcomes in the Dietary Intervention Stem Cells and Colorectal Cancer (DISC) Study.The DISC Study was a double-blind, randomised controlled trial that supplemented healthy participants with RS and/or PD or placebo for 50 days in a 2*2 factorial design. DNA was extracted from stool samples collected pre- and post-intervention, and V4 16S rRNA gene sequencing was used to profile the gut microbiota. Metabolite concentrations were measured in stool, plasma, and urine by high-performance liquid chromatography.A total of 58 participants with paired samples available were included. After 50 days, no effects of RS or PD were detected on composition of the gut microbiota diversity (alpha- and beta-diversity), on genus relative abundance, or on metabolite concentrations. However, Drichlet's multinomial mixture clustering-based approach suggests that some participants changed microbial enterotype post-intervention.The gut microbiome and faecal, plasma, and urinary microbial metabolites were stable in response to a 50-day fibre intervention in middle aged adults. Larger and longer studies, including those which explore the effects of specific fibre sub-types, may be required to determine the relationships between fibre intake, the gut microbiome, and host-health.

A novel dynamic proteomics approach for the measurement of broiler chicken protein fractional synthesis rate.

RATIONALE: The study of protein synthesis in farm animals is uncommon despite its potential to increase knowledge about metabolism and discover new biomarkers of health and growth status. The present study describes a novel dynamic proteomics approach for the measurement of protein fractional synthesis rate (FSR) in broiler chickens. METHODS: Chickens received a 10 g/kg oral dose of 2 H2 O at day 21 of their life. Body water 2 H abundance was measured in plasma samples using a portable Fourier transform infrared spectrometer. Free and protein-bound amino acids (AAs) were isolated and had their 2 H enrichment measured by gas chromatography with mass spectrometry (GC/MS). Peptide 2 H enrichment was measured by proteomics analysis of plasma and muscle samples. Albumin, fibrinogen and muscle protein FSR were calculated from GC/MS and proteomics data. RESULTS: Ala appeared to be more enriched at the site of protein synthesis than in the AA free pools. Glu was found to be the AA closest to isotopic equilibrium between the different AA pools. Glu was used as an anchor to calculate n(AA) values necessary for chicken protein FSR calculation in dynamic proteomics studies. FSR values calculated using proteomics data and GC/MS data showed good agreement as evidenced by a Bland-Altman residual plot. CONCLUSIONS: A new dynamic proteomics approach for the measurement of broiler chicken individual protein FSR based on the administration of a single 2 H2 O oral bolus has been developed and validated. The proposed approach could facilitate new immunological and nutritional studies on free-living animals.

Understanding the role of the gut in undernutrition: what can technology tell us?

Gut function remains largely underinvestigated in undernutrition, despite its critical role in essential nutrient digestion, absorption and assimilation. In areas of high enteropathogen burden, alterations in gut barrier function and subsequent inflammatory effects are observable but remain poorly characterised. Environmental enteropathy (EE)-a condition that affects both gut morphology and function and is characterised by blunted villi, inflammation and increased permeability-is thought to play a role in impaired linear growth (stunting) and severe acute malnutrition. However, the lack of tools to quantitatively characterise gut functional capacity has hampered both our understanding of gut pathogenesis in undernutrition and evaluation of gut-targeted therapies to accelerate nutritional recovery. Here we survey the technology landscape for potential solutions to improve assessment of gut function, focussing on devices that could be deployed at point-of-care in low-income and middle-income countries (LMICs). We assess the potential for technological innovation to assess gut morphology, function, barrier integrity and immune response in undernutrition, and highlight the approaches that are currently most suitable for deployment and development. This article focuses on EE and undernutrition in LMICs, but many of these technologies may also become useful in monitoring of other gut pathologies.

Dietary supplementation with inulin-propionate ester or inulin improves insulin sensitivity in adults with overweight and obesity with distinct effects on the gut microbiota, plasma metabolome and systemic inflammatory responses: a randomised cross-over trial.

OBJECTIVE: To investigate the underlying mechanisms behind changes in glucose homeostasis with delivery of propionate to the human colon by comprehensive and coordinated analysis of gut bacterial composition, plasma metabolome and immune responses. DESIGN: Twelve non-diabetic adults with overweight and obesity received 20 g/day of inulin-propionate ester (IPE), designed to selectively deliver propionate to the colon, a high-fermentable fibre control (inulin) and a low-fermentable fibre control (cellulose) in a randomised, double-blind, placebo-controlled, cross-over design. Outcome measurements of metabolic responses, inflammatory markers and gut bacterial composition were analysed at the end of each 42-day supplementation period. RESULTS: Both IPE and inulin supplementation improved insulin resistance compared with cellulose supplementation, measured by homeostatic model assessment 2 (mean±SEM 1.23±0.17 IPE vs 1.59±0.17 cellulose, p=0.001; 1.17±0.15 inulin vs 1.59±0.17 cellulose, p=0.009), with no differences between IPE and inulin (p=0.272). Fasting insulin was only associated positively with plasma tyrosine and negatively with plasma glycine following inulin supplementation. IPE supplementation decreased proinflammatory interleukin-8 levels compared with cellulose, while inulin had no impact on the systemic inflammatory markers studied. Inulin promoted changes in gut bacterial populations at the class level (increased Actinobacteria and decreased Clostridia) and order level (decreased Clostridiales) compared with cellulose, with small differences at the species level observed between IPE and cellulose. CONCLUSION: These data demonstrate a distinctive physiological impact of raising colonic propionate delivery in humans, as improvements in insulin sensitivity promoted by IPE and inulin were accompanied with different effects on the plasma metabolome, gut bacterial populations and markers of systemic inflammation.

The effects of dietary supplementation with inulin and inulin-propionate ester on hepatic steatosis in adults with non-alcoholic fatty liver disease.

The short chain fatty acid (SCFA) propionate, produced through fermentation of dietary fibre by the gut microbiota, has been shown to alter hepatic metabolic processes that reduce lipid storage. We aimed to investigate the impact of raising colonic propionate production on hepatic steatosis in adults with non-alcoholic fatty liver disease (NAFLD). Eighteen adults were randomized to receive 20 g/d of an inulin-propionate ester (IPE), designed to deliver propionate to the colon, or an inulin control for 42 days in a parallel design. The change in intrahepatocellular lipid (IHCL) following the supplementation period was not different between the groups (P = 0.082), however, IHCL significantly increased within the inulin-control group (20.9% ± 2.9% to 26.8% ± 3.9%; P = 0.012; n = 9), which was not observed within the IPE group (22.6% ± 6.9% to 23.5% ± 6.8%; P = 0.635; n = 9). The predominant SCFA from colonic fermentation of inulin is acetate, which, in a background of NAFLD and a hepatic metabolic profile that promotes fat accretion, may provide surplus lipogenic substrate to the liver. The increased colonic delivery of propionate from IPE appears to attenuate this acetate-mediated increase in IHCL.

Systemic availability and metabolism of colonic-derived short-chain fatty acids in healthy subjects: a stable isotope study.

KEY POINTS: The short-chain fatty acids (SCFAs) are bacterial metabolites produced during the colonic fermentation of undigested carbohydrates, such as dietary fibre and prebiotics, and can mediate the interaction between the diet, the microbiota and the host. We quantified the fraction of colonic administered SCFAs that could be recovered in the systemic circulation, the fraction that was excreted via the breath and urine, and the fraction that was used as a precursor for glucose, cholesterol and fatty acids. This information is essential for understanding the molecular mechanisms by which SCFAs beneficially affect physiological functions such as glucose and lipid metabolism and immune function. ABSTRACT: The short-chain fatty acids (SCFAs), acetate, propionate and butyrate, are bacterial metabolites that mediate the interaction between the diet, the microbiota and the host. In the present study, the systemic availability of SCFAs and their incorporation into biologically relevant molecules was quantified. Known amounts of 13 C-labelled acetate, propionate and butyrate were introduced in the colon of 12 healthy subjects using colon delivery capsules and plasma levels of 13 C-SCFAs 13 C-glucose, 13 C-cholesterol and 13 C-fatty acids were measured. The butyrate-producing capacity of the intestinal microbiota was also quantified. Systemic availability of colonic-administered acetate, propionate and butyrate was 36%, 9% and 2%, respectively. Conversion of acetate into butyrate (24%) was the most prevalent interconversion by the colonic microbiota and was not related to the butyrate-producing capacity in the faecal samples. Less than 1% of administered acetate was incorporated into cholesterol and <15% in fatty acids. On average, 6% of colonic propionate was incorporated into glucose. The SCFAs were mainly excreted via the lungs after oxidation to 13 CO2 , whereas less than 0.05% of the SCFAs were excreted into urine. These results will allow future evaluation and quantification of SCFA production from 13 C-labelled fibres in the human colon by measurement of 13 C-labelled SCFA concentrations in blood.

The diet-derived short chain fatty acid propionate improves beta-cell function in humans and stimulates insulin secretion from human islets in vitro.

AIMS: Diet-derived short chain fatty acids (SCFAs) improve glucose homeostasis in vivo, but the role of individual SCFAs and their mechanisms of action have not been defined. This study evaluated the effects of increasing colonic delivery of the SCFA propionate on ß-cell function in humans and the direct effects of propionate on isolated human islets in vitro. MATERIALS AND METHODS: For 24 weeks human subjects ingested an inulin-propionate ester that delivers propionate to the colon. Acute insulin, GLP-1 and non-esterified fatty acid (NEFA) levels were quantified pre- and post-supplementation in response to a mixed meal test. Expression of the SCFA receptor FFAR2 in human islets was determined by western blotting and immunohistochemistry. Dynamic insulin secretion from perifused human islets was quantified by radioimmunoassay and islet apoptosis was determined by quantification of caspase 3/7 activities. RESULTS: Colonic propionate delivery in vivo was associated with improved ß-cell function with increased insulin secretion that was independent of changes in GLP-1 levels. Human islet ß-cells expressed FFAR2 and propionate potentiated dynamic glucose-stimulated insulin secretion in vitro, an effect that was dependent on signalling via protein kinase C. Propionate also protected human islets from apoptosis induced by the NEFA sodium palmitate and inflammatory cytokines. CONCLUSIONS: Our results indicate that propionate has beneficial effects on ß-cell function in vivo, and in vitro analyses demonstrated that it has direct effects to potentiate glucose-stimulated insulin release and maintain ß-cell mass through inhibition of apoptosis. These observations support ingestion of propiogenic dietary fibres to maintain healthy glucose homeostasis.

Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults.

OBJECTIVE: The colonic microbiota ferment dietary fibres, producing short chain fatty acids. Recent evidence suggests that the short chain fatty acid propionate may play an important role in appetite regulation. We hypothesised that colonic delivery of propionate would increase peptide YY (PYY) and glucagon like peptide-1 (GLP-1) secretion in humans, and reduce energy intake and weight gain in overweight adults. DESIGN: To investigate whether propionate promotes PYY and GLP-1 secretion, a primary cultured human colonic cell model was developed. To deliver propionate specifically to the colon, we developed a novel inulin-propionate ester. An acute randomised, controlled cross-over study was used to assess the effects of this inulin-propionate ester on energy intake and plasma PYY and GLP-1 concentrations. The long-term effects of inulin-propionate ester on weight gain were subsequently assessed in a randomised, controlled 24-week study involving 60 overweight adults. RESULTS: Propionate significantly stimulated the release of PYY and GLP-1 from human colonic cells. Acute ingestion of 10 g inulin-propionate ester significantly increased postprandial plasma PYY and GLP-1 and reduced energy intake. Over 24 weeks, 10 g/day inulin-propionate ester supplementation significantly reduced weight gain, intra-abdominal adipose tissue distribution, intrahepatocellular lipid content and prevented the deterioration in insulin sensitivity observed in the inulin-control group. CONCLUSIONS: These data demonstrate for the first time that increasing colonic propionate prevents weight gain in overweight adult humans. TRIAL REGISTRATION NUMBER: NCT00750438.

Total body water measurement using the 2H dilution technique for the assessment of body composition of Kuwaiti children.

OBJECTIVE: The 2H dilution technique is the reference method to estimate total body water for body composition assessment. The aims of the present study were to establish the total body water technique at the Kuwait Institute for Scientific Research and assess body composition of Kuwaiti children. DESIGN: The isotope ratio mass spectrometer was calibrated with defined international reference water standards. A non-random sampling approach was used to recruit a convenience sample of Kuwaiti children. A dose of 2H2O, 1-3 g, was consumed after an overnight fast and 2H enrichment in baseline and post-dose urine samples was measured. Total body water was calculated and used to estimate fat-free mass. Fat mass was estimated as body weight minus fat-free mass. SETTING: The total body water study was implemented in primary schools. SUBJECTS: Seventy-five boys and eighty-three girls (7-9 years). RESULTS: Measurements of the isotope ratio mass spectrometer were confirmed to be accurate and precise. Children were classified as normal weight, overweight or obese according to the WHO based on BMI-for-age Z-scores. Normal-weight and overweight girls had significantly higher percentage body fat (median (range): 32·4 % (24·7-39·3 %) and 38·3 % (29·3-44·2 %), respectively) compared with boys (median (range): 26·5 % (14·2-37·1 %) and 34·6 % (29·9-40·2 %), respectively). No gender difference was found in obese children (median 46·5 % v. 45·6 %). CONCLUSIONS: The establishment of a state-of-the-art stable isotope laboratory for assessment of body composition provides an opportunity to explore a wide range of applications to better understand the relationship between body size, body composition and risk of developing non-communicable diseases in Kuwait.

Existing and emerging technologies for measuring stable isotope labelled retinol in biological samples: isotope dilution analysis of body retinol stores.

This paper discusses some of the recent improvements in instrumentation used for stable isotope tracer measurements in the context of measuring retinol stores, in vivo. Tracer costs, together with concerns that larger tracer doses may perturb the parameter under study, demand that ever more sensitive mass spectrometric techniques are developed. GCMS is the most widely used technique. It has high sensitivity in terms of sample amount and uses high resolution GC, yet its ability to detect low isotope ratios is limited by background noise. LCMSMS may become more accessible for tracer studies. Its ability to measure low level stable isotope tracers may prove superior to GCMS, but it is isotope ratio MS (IRMS) that has been designed specifically for low level stable isotope analysis through accurate analysis of tracer:tracee ratios (the tracee being the unlabelled species). Compound-specific isotope analysis, where GC is interfaced to IRMS, is gaining popularity. Here, individual 13C-labelled compounds are separated by GC, combusted to CO2 and transferred on-line for ratiometric analysis by IRMS at the ppm level. However, commercially-available 13C-labelled retinol tracers are 2 - 4 times more expensive than deuterated tracers. For 2H-labelled compounds, GC-pyrolysis-IRMS has now become more generally available as an operating mode on the same IRMS instrument. Here, individual compounds are separated by GC and pyrolysed to H2 at high temperature for analysis by IRMS. It is predicted that GC-pyrolysis-IRMS will facilitate low level tracer procedures to measure body retinol stores, as has been accomplished in the case of fatty acids and amino acids. Sample size requirements for GC-P-IRMS may exceed those of GCMS, but this paper discusses sample preparation procedures and predicts improvements, particularly in the efficiency of sample introduction.

Effect of an immune challenge and two feed supplements on broiler chicken individual breast muscle protein synthesis rate.

Optimization of broiler chicken breast muscle protein accretion is key for the efficient production of poultry meat, whose demand is steadily increasing. In a context where antimicrobial growth promoters use is being restricted, it is important to find alternatives as well as to characterize the effect of immunological stress on broiler chicken's growth. Despite its importance, research on broiler chicken muscle protein dynamics has mostly been limited to the study of mixed protein turnover. The present study aims to characterize the effect of a bacterial challenge and the feed supplementation of citrus and cucumber extracts on broiler chicken individual breast muscle proteins fractional synthesis rates (FSR) using a recently developed dynamic proteomics pipeline. Twenty-one day-old broiler chickens were administered a single 2H2O dose before being culled at different timepoints. A total of 60 breast muscle protein extracts from five experimental groups (Unchallenged, Challenged, Control Diet, Diet 1 and Diet 2) were analysed using a DDA proteomics approach. Proteomics data was filtered in order to reliably calculate multiple proteins FSR making use of a newly developed bioinformatics pipeline. Broiler breast muscle proteins FSR uniformly decreased following a bacterial challenge, this change was judged significant for 15 individual proteins, the two major functional clusters identified as well as for mixed breast muscle protein. Citrus or cucumber extract feed supplementation did not show any effect on the breast muscle protein FSR of immunologically challenged broilers. The present study has identified potential predictive markers of breast muscle growth and provided new information on broiler chicken breast muscle protein synthesis which could be essential for improving the efficiency of broiler chicken meat production. SIGNIFICANCE: The present study constitutes the first dynamic proteomics study conducted in a farm animal species which has characterized FSR in a large number of proteins, establishing a precedent for biomarker discovery and assessment of health and growth status. Moreover, it has been evidenced that the decrease in broiler chicken breast muscle protein following an immune challenge is a coordinated event which seems to be the main cause of the decreased growth observed in these animals.

Increase in colonic PRopionate as a method of prEVENTing weight gain over 12 months in adults aged 20-40 years (iPREVENT): a multi-centre, double-blind, randomised, parallel-group trial.

Background: Obesity drives metabolic disease development. Preventing weight gain during early adulthood could mitigate later-life chronic disease risk. Increased dietary fibre intake, leading to enhanced colonic microbial fermentation and short-chain fatty acid (SCFA) production, is associated with lower body weight. Despite national food policy recommendations to consume 30 g of dietary fibre daily, only 9% of adults achieve this target. Inulin-propionate ester (IPE) selectively increases the production of the SCFA propionate in the colon. In previous studies, IPE has prevented weight gain in middle-aged adults over 6 months, compared with the inulin control. IPE is a novel food ingredient that can be added to various commonly consumed foods with a potential health benefit. This 12-month study aimed to determine whether using IPE to increase colonic propionate prevents further weight gain in overweight younger adults. Methods: This multi-centre randomised-controlled, double-blind trial was conducted in London and Glasgow, UK. Recruited participants were individuals at risk of weight gain, aged between 20 and 40 years and had an overweight body mass index. Sealed Envelope Software was used to randomise participants to consume 10 g of IPE or inulin (control), once per day for 12 months. The primary outcome was the weight gained from baseline to 12 months, analysed by an 'Intention to Treat' strategy. The safety profile and tolerability of IPE were monitored through adverse events and compliance. This study is registered with the International Standard Randomised Controlled Trials (ISRCT) Database (ISRCT number: 16299902). Findings: Participants (n = 135 per study arm) were recruited from July 2019 to October 2021. At 12 months, there was no significant difference in baseline-adjusted mean weight gain for IPE compared with control (1.02 kg, 95% CI: -0.37 to 2.41; p = 0.15; n = 226). Neither the IPE (+1.22 kg) nor the control arm (+0.07 kg) unadjusted mean gains in body weight reached the expected 2 kg threshold. In the IPE arm, fat-free mass was greater by 1.07 kg (95% CI: 0.21-1.93), and blood glucose elevated by 0.11 mmol/L (95% CI: 0.01-0.21). Compliance, determined by intake of ≥50% sachets, was reached by 63% of IPE participants. There were no unexpected adverse events or safety concerns. Interpretation: Our study indicates that at 12 months, IPE did not differentially affect weight gain, compared with the inulin control, in adults between 20 and 40 years of age, at risk of obesity. Funding: NIHR EME Programme (15/185/16).

Intrinsic labelling of common beans with 2H2O to enable estimates of protein digestibility.

BACKGROUND: The use of plant protein intrinsically labelled with stable isotopes provides an innovative solution to assess the efficiency of protein intake by humans. Here, the incorporation of 2H has been applied to intrinsically labelled plant protein in the common bean. This study aimed to evaluate which is the best phenological phase of seed maturation to incorporate the heavy hydrogen isotope 2H into seed amino acids. Common beans (Phaseolus vulgaris L.) were grown in pots, then, after 50 days sowing, 2H2O dissolved in irrigation water was applied, then again at an interval of either 3, 6, 9, and 12 days. RESULTS: Applications of 2H2O at 6, 9, and 12 days after the first application, in the full-flowering stage, were the best treatments for enriching protein-bound amino acids in the bean seed with 2H. CONCLUSION: All treatments resulted in enrichment above 500 ppm, so the treatments (quantity and timing of 2H2O addition) were deemed successful for enriching bean seeds. This makes the intrinsically labelled seeds suitable for preparing test meals to assess the digestion and essential amino acid absorption of common bean amino acids in human subjects.

Effects of variations in atmospheric temperature and humidity on the estimation of exclusive breastfeeding status using the deuterium oxide dose-to-mother technique.

Background: The deuterium dose-to-mother (DTM) method measures the human milk intake of breastfed children. Recently, the use of this method has been expanded to classify babies objectively as exclusively breast fed (EBF) or not (non-EBF) based on quantification of non-milk oral water intake (NMOI). However, the calculation of NMOI estimates involves atmospheric temperature and humidity. Objective: To evaluate the effects of atmospheric temperature and humidity on NMOI calculation and the classification of exclusive breastfeeding. Methods: The effect of indoor temperature and relative humidity on NMOI and the estimated prevalence of non-EBF were examined in two existing data sets of DTM in children by varying temperature in the range of 15 to 35°C and relative humidity in the range of 20 to 80% representing the maximum span of indoor conditions expected. Population-level estimates of NMOI distributions were derived using the rstan package for R v2.21.2. Results: The NMOI decreased at a rate of -1.15 g/day per °C increase and at a rate of -1.01 g/day per percent increase in relative humidity; this was due to variations in non-oral water intake from the atmosphere, a component of the calculation of NMOI, which is dependent on temperature and humidity. For the various locations considered, the mean calculated NMOI varied between 24.6 and 53.3 g/day using the same input data. In the mixed-fed sample of babies, the prevalence of non-EBF based on the earlier defined NMOI cut-off of 86.6 g/day was reduced by 19% when relative humidity was increased by 60%. Conclusions: Atmospheric conditions are essential factors in the computation of NMOI, used in the objective classification of babies as exclusively breast fed or not, and should be considered when the DTM method is used to classify exclusive breastfeeding.

The glycemic response does not reflect the in vivo starch digestibility of fiber-rich wheat products in healthy men.

Starchy food products differ in the rate of starch digestion, which can affect their metabolic impact. In this study, we examined how the in vivo starch digestibility is reflected by the glycemic response, because this response is often used to predict starch digestibility. Ten healthy male volunteers [age 21 ± 0.5 y, BMI 23 ± 0.6 kg/m² (mean ± SEM)] participated in a cross-over study, receiving three different meals: pasta with normal wheat bran (PA) and bread with normal (CB) or purple wheat bran (PBB). Purple wheat bran was added in an attempt to decrease the rate of starch digestion. The meals were enriched in ¹³C and the dual isotope technique was applied to calculate the rate of appearance of exogenous glucose (RaE). The ¹³C-isotopic enrichment of glucose in plasma was measured with GC/combustion/isotope ratio MS (IRMS) and liquid chromatography/IRMS. Both IRMS techniques gave similar results. Plasma glucose concentrations [2-h incremental AUC (iAUC)] did not differ between the test meals. The RaE was similar after consumption of CB and PBB, showing that purple wheat bran in bread does not affect in vivo starch digestibility. However, the iAUC of RaE after men consumed PA was less than after they consumed CB (P < 0.0001) despite the similar glucose response. To conclude, the glycemic response does not always reflect the in vivo starch digestibility. This could have implications for intervention studies in which the glycemic response is used to characterize test products.

Muscle protein synthesis and muscle/metabolic responses to resistance exercise training in South Asian and White European men.

The aims of the current study, therefore, were to compare (1) free-living MPS and (2) muscle and metabolic adaptations to resistance exercise in South Asian and white European adults. Eighteen South Asian and 16 White European men were enrolled in the study. Free-living muscle protein synthesis was measured at baseline. Muscle strength, body composition, resting metabolic rate, VO2max and metabolic responses (insulin sensitivity) to a mixed meal were measured at baseline and following 12 weeks of resistance exercise training. Free-living muscle protein synthesis was not different between South Asians (1.48 ± 0.09%/day) and White Europeans (1.59 ± 0.15%/day) (p = 0.522). In response to resistance exercise training there were no differences, between South Asians and White Europeans, muscle mass, lower body strength or insulin sensitivity. However, there were differences between the ethnicities in response to resistance exercise training in body fat, resting carbohydrate and fat metabolism, blood pressure, VO2max and upper body strength with responses less favourable in South Asians. In this exploratory study there were no differences in muscle protein synthesis or anabolic and metabolic responses to resistance exercise, yet there were less favourable responses in several outcomes. These findings require further investigation.

Strong anion exchange liquid chromatographic separation of protein amino acids for natural 13C-abundance determination by isotope ratio mass spectrometry.

Amino acids are the building blocks of proteins and the analysis of their (13)C abundances is greatly simplified by the use of liquid chromatography (LC) systems coupled with isotope ratio mass spectrometry (IRMS) compared with gas chromatography (GC)-based methods. To date, various cation exchange chromatography columns have been employed for amino acid separation. Here, we report strong anion exchange chromatography (SAX) coupled to IRMS with a Liquiface interface for amino acid δ(13)C determination. Mixtures of underivatised amino acids (0.1-0.5 mM) and hydrolysates of representative proteins (prawns and bovine serum albumin) were resolved by LC/IRMS using a SAX column and inorganic eluents. Background inorganic carbon content was minimised through careful preparation of alkaline reagents and use of a pre-injector on-line carbonate removal device. SAX chromatography completely resolved 11 of the 16 expected protein amino acids following acid hydrolysis in underivatised form. Basic and neutral amino acids were resolved with 35 mM NaOH in isocratic mode. Elution of the aromatic and acidic amino acids required a higher hydroxide concentration (180 mM) and a counterion (NO 3-, 5-25 mM). The total run time was 70 min. The average δ(13)C precision of baseline-resolved peaks was 0.75‰ (range 0.04 to 1.06‰). SAX is a viable alternative to cation chromatography, especially where analysis of basic amino acids is important. The technology shows promise for (13)C amino acid analysis in ecology, archaeology, forensic science, nutrition and protein metabolism.

Quantitation of plasma 13C-galactose and 13C-glucose during exercise by liquid chromatography/isotope ratio mass spectrometry.

The utilisation of carbohydrate sources under exercise conditions is of considerable importance in performance sports. Incorporation of optimal profiles of macronutrients can improve endurance performance in athletes. However, gaining an understanding of the metabolic partitioning under sustained exercise can be problematical and isotope labelling approaches can help quantify substrate utilisation. The utilisation of oral galactose was investigated using (13)C-galactose and measurement of plasma galactose and glucose enrichment by liquid chromatography/isotope ratio mass spectrometry (LC/IRMS). As little as 100 µL plasma could readily be analysed with only minimal sample processing. Fucose was used as a chemical and isotopic internal standard for the quantitation of plasma galactose and glucose concentrations, and isotopic enrichment. The close elution of galactose and glucose required a correction routine to be implemented to allow the measurement, and correction, of plasma glucose δ(13)C, even in the presence of very highly enriched galactose. A Bland-Altman plot of glucose concentration measured by LC/IRMS against glucose measured by an enzymatic method showed good agreement between the methods. Data from seven trained cyclists, undergoing galactose supplementation before exercise, demonstrate that galactose is converted into glucose and is available for subsequent energy metabolism.

Safety and toxicological evaluation of a synthetic vitamin K2, menaquinone-7.

Menaquinone-7 (MK-7) is part of a family of vitamin K that are essential co-factors for the enzyme γ-glutamyl carboxylase, which is involved in the activation of γ-carboxy glutamate (Gla) proteins in the body. Gla proteins are important for normal blood coagulation and normality of bones and arteries. The objective of this study was to examine the potential toxicity of synthetic MK-7 in BomTac:NMRI mice and in Sprague-Dawley rats. In an acute oral toxicity test, mice were administered a single oral dose of 2000 mg/kg body weight (limit dose) and no toxicity was observed during the 14-day observation period. In the subchronic oral toxicity test in rats, animals were administered MK-7 for 90 days by gavage at the following doses: 0 (vehicle control, corn oil), 2.5, 5, and 10 mg/kg body weight/day. All generated data, including clinical observations, ophthalmology, clinical pathology, gross necropsy, and histopathology, revealed no compound-related toxicity in rats. Any statistically significant findings in clinical pathology parameters and/or organ weights noted were considered to be within normal biological variability. Therefore, under the conditions of this experiment, the median lethal dose (LD(50)) of MK-7 after a single oral administration in mice was determined to be greater than the limit dose level of 2000 mg/kg body weight. The no observed adverse effect level (NOAEL) of MK-7, when administered orally to rats for 90 days, was considered to be equal to 10 mg/kg body weight/day, the highest dose tested, based on lack of toxicity during the 90-day study period.

Mechanisms of obesity in children and adults with phenylketonuria on contemporary treatment.

BACKGROUND & AIMS: Obesity prevalence in people with phenylketonuria (PKU) is comparable to that of the general population but the underlying aetiology remains unknown. To assess body composition, dietary intake, moderate physical activity duration (MPAD) and energy expenditure (MPAEE), resting metabolic rate (RMR), diet-induced thermogenesis (DIT), fasting and postprandial fat (FOx) and carbohydrate oxidation (CHOOx), in PKU people and healthy Controls. METHODS: Participants were PKU people (n = 16) and healthy controls (n = 15). Body composition was measured with stable isotopes using deuterium as tracer, dietary intake from 4-day food diaries, MPAD and MPAEE from 7-day activity counts measured by triaxial accelerometers, calibrated against individual rates of oxygen consumption and carbon dioxide production, RMR, DIT, FOx and CHOOx by indirect calorimetry. RESULTS: Body composition, DIT, FOx, CHOOx and RMR did not differ between the PKU and the Control groups. MPAD (PKU, 73 ± 26 min/week; Control, 152 ± 43 min/week) and MPAEE (PKU, 404 ± 127 kcal/week; Control, 741 ± 153 kcal/week) were lower (P < 0.05) in the PKU than the Control group. Raised phenylalanine levels were inversely related with MPAD and MPAEE. Energy intake and energy provided by protein did not differ between the groups, while energy proportion obtained from carbohydrate was higher (PKU, 60 ± 2%; Control, 51 ± 2%; P < 0.05) and from fat lower (PKU, 24 ± 2%; Control, 35 ± 3%; P < 0.05) in the PKU than in the Control group. CONCLUSION: People with PKU spent less time and expend less energy in moderate physical activity and have a higher intake of energy from CHO which may be involved in the underlying mechanisms of obesity in PKU.