Dietary fibre in Europe: current state of knowledge on definitions, sources, recommendations, intakes and relationships to health.

Research into the analysis, physical properties and health effects of dietary fibre has continued steadily over the last 40-50 years. From the knowledge gained, countries have developed guidelines for their populations on the optimal amount of fibre to be consumed each day. Food composition tables from many countries now contain values for the dietary fibre content of foods, and, from these, combined with dietary surveys, population intakes have been determined. The present review assessed the uniformity of the analytical methods used, health claims permitted, recommendations and intakes, particularly from national surveys across Europe and around the world. It also assessed current knowledge on health effects of dietary fibre and related the impact of different fibre types on health. The overall intent was to be able to provide more detailed guidance on the types of fibre which should be consumed for good health, rather than simply a total intake figure, the current situation. Analysis of data indicated a fair degree of uniformity in the definition of dietary fibre, the method used for analysis, the recommended amount to be consumed and a growing literature on effects on digestive health and disease risk. However, national dietary survey data showed that intakes do not reach recommendations and very few countries provide guidance on the types of fibre that are preferable to achieve recommended intakes. Research gaps were identified and ideas suggested to provide information for more detailed advice to the public about specific food sources that should be consumed to achieve health benefits.

Impact of perinatal prebiotic consumption on gestating mice and their offspring: a preliminary report.

To assess the impact of prebiotic supplementation during gestation and fetal and early neonatal life, gestating BALB/cj dam mice were fed either a control or a prebiotic (galacto-oligosaccharides-inulin, 9:1 ratio)-enriched diet throughout pregnancy and lactation, and allowed to nurse their pups until weaning. At the time of weaning, male offspring mice were separated from their mothers, weaned to the same solid diet as their dam and their growth was monitored until killed 48 d after weaning. Prebiotic treatment affected neither the body-weight gain nor the food intake of pregnant mice. In contrast, at the time of weaning, pups that had been nursed by prebiotic-fed dams had a higher body weight (11.0 (se 1.2) g) than pups born from control dams (9.8 (se 0.9) g). At 48 d after weaning, significantly higher values were observed for colon length and muscle mass in the offspring of prebiotic-fed dams (1.2 (se 0.1) cm/cm and 5.7 (se 1.8) mg/g, respectively), compared with control offspring (1.1 (se 0.1) cm/cm and 2.9 (se 0.9) mg/g, respectively), without any difference in spleen and stomach weight, or serum leptin concentration. The present preliminary study suggests that altering the fibre content of the maternal diet during both pregnancy and lactation enhances offspring growth, through an effect on intestinal and muscle mass rather than fat mass accretion.

Determination of total dietary fiber (CODEX definition) by enzymatic-gravimetric method and liquid chromatography: collaborative study.

A method for the determination of total dietary fiber (TDF), as defined by the CODEX Alimentarius, was validated in foods. Based upon the principles of AOAC Official Methods 985.29, 991.43, 2001.03, and 2002.02, the method quantitates high- and low-molecular-weight dietary fiber (HMWDF and LMWDF, respectively). In 2007, McCleary described a method of extended enzymatic digestion at 37 degrees C to simulate human intestinal digestion followed by gravimetric isolation and quantitation of HMWDF and the use of LC to quantitate low-molecular-weight soluble dietary fiber (LMWSDF). The method thus quantitates the complete range of dietary fiber components from resistant starch (by utilizing the digestion conditions of AOAC Method 2002.02) to digestion resistant oligosaccharides (by incorporating the deionization and LC procedures of AOAC Method 2001.03). The method was evaluated through an AOAC collaborative study. Eighteen laboratories participated with 16 laboratories returning valid assay data for 16 test portions (eight blind duplicates) consisting of samples with a range of traditional dietary fiber, resistant starch, and nondigestible oligosaccharides. The dietary fiber content of the eight test pairs ranged from 11.57 to 47.83%. Digestion of samples under the conditions of AOAC Method 2002.02 followed by the isolation and gravimetric procedures of AOAC Methods 985.29 and 991.43 results in quantitation of HMWDF. The filtrate from the quantitation of HMWDF is concentrated, deionized, concentrated again, and analyzed by LC to determine the LMWSDF, i.e., all nondigestible oligosaccharides of degree of polymerization > or =3. TDF is calculated as the sum of HMWDF and LMWSDF. Repeatability standard deviations (Sr) ranged from 0.41 to 1.43, and reproducibility standard deviations (S(R)) ranged from 1.18 to 5.44. These results are comparable to other official dietary fiber methods, and the method is recommended for adoption as Official First Action.

Arabinoxylan content and grain tissue distribution are good predictors of the dietary fibre content and their nutritional properties in wheat products.

Wheat millstreams and wheat-based foods (pasta, biscuits and bread) enriched or not in dietary fibre with fractions extracted from wheat grains, have been characterized either for their total dietary fibre content (TDF) and their arabinoxylan (AX) content. A strong correlation (r2 = 0.98) is observed between the AX and TDF contents indicating that AX can be used to estimate TDF content in wheat products. Moreover, by adding a previous step including enzymatic hydrolysis with a xylanase, a functional evaluation of DF is proposed based on the amount of AX released by the enzyme. Xylanase hydrolysable AX are likely also released by microbiota's enzymes in the gut and therefore an indicator for the proportion of fermentable DF in grain fractions and wheat-based foods (pasta, biscuits and bread). This assay opens the door for simple characterization of qualitative attribute of cereal DF.

Functional food for pregnant, lactating women and in perinatal nutrition: a role for dietary fibres?

PURPOSE OF REVIEW: If the benefits of dietary fibre in healthy adults have extensively been studied, little information is available on the specific needs of pregnant, lactating women or foetus. As far as infants are concerned, milk oligosaccharides are supposed to be the optimal 'dietary fibre'. The supplementation of infant formula with prebiotic oligosaccharides is still discussed. However, recent studies provide a large amount of information, allowing a new discussion on this topic. RECENT FINDINGS: Most recent findings are linked to the involvement of dietary fibre in occurrence or prevention of obesity. The multiple mechanisms appear more clearly than earlier. This finding will soon allow appropriate counselling for young mothers at risk of obesity and/or postpartum retention weight, gestational diabetes and preeclampsia. Another area which benefits from recent research is the use of prebiotics in formula. SUMMARY: Pregnancy is a critical period during which many physiologic changes occurred and is associated with several gut disorders and metabolic diseases. Dietary fibre may be helpful in the prevention and management of these diseases. Lactation and pregnancy are two phases during which food consumption of the mother can interact with the physiology of the baby. Moreover, the use of formula supplemented in oligosaccharides is able to compensate for the lack of some of the complex molecules naturally present in human milk.

French Recommendations for Sugar Intake in Adults: A Novel Approach Chosen by ANSES.

This article presents a systematic review of the scientific evidence linking sugar consumption and health in the adult population performed by a group of experts, mandated by the French Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement, et du travail (ANSES). A literature search was performed by crossing search terms for overweight/obesity, diabetes/insulin resistance, dyslipidemia/cardiovascular diseases, non-alcoholic fatty liver diseases (NAFLD), and uric acid concentrations on one hand and for intake of sugars on the other. Controlled mechanistic studies, prospective cohort studies, and randomized clinical trials were extracted and assessed. A literature analysis supported links between sugar intake and both total energy intake and body weight gain, and between sugar intake and blood triglycerides independently of total energy intake. The effects of sugar on blood triglycerides were shown to be mediated by the fructose component of sucrose and were observed with an intake of fructose >50 g/day. In addition, prospective cohort studies showed associations between sugar intake and the risk of diabetes/insulin resistance, cardiovascular diseases, NAFLD, and hyperuricemia. Based on these observations, ANSES proposed to set a maximum limit to the intake of total sugars containing fructose (sucrose, glucose⁻fructose syrups, honey or other syrups, and natural concentrates, etc.) of 100 g/day.

Effects of high-resistant-starch banana flour (RS(2)) on in vitro fermentation and the small-bowel excretion of energy, nutrients, and sterols: an ileostomy study.

BACKGROUND: Resistant starch (RS) has attracted interest because of its effects in the colon and implications for health. Knowledge of how RS influences small-intestinal absorption of nutrients, sterol metabolism, and colonic fermentation is sparse. OBJECTIVES: The objectives of this study were to measure the effect of RS(2), a type of RS in banana flour, on the ileal excretion of energy, nutrients, and sterols, and to compare in vivo measurement of RS in the ileostomy model with previously published intubation data. In addition, we sought to estimate a fermentation pattern by using ileal effluents for in vitro fermentation. DESIGN: The present study was divided into 2 parts. Study A involved 10 ileostomy subjects who were given a controlled diet with the addition of 30 g raw green banana flour (RBF)/d, which contains RS(2), or cooked green banana flour (CBF)/d in random order. Study B involved 7 ileostomy subjects who were given a plant-polysaccharide-free diet with the addition of 30 g RBF/d. RESULTS: In study A, the dry weight of the ileostomy effluents and the ileal excretion of energy, iron, and chenodeoxycholic acid, but not total sterols, were higher after the addition of RBF than of CBF to the diet. In vitro fermentation of the ileal effluents obtained after the addition of RBF to the diet showed higher concentrations of acetate and butyrate. In study B, the ileal excretion of starch was lower than the amount calculated from earlier studies by use of the intubation technique. CONCLUSIONS: The addition of RBF containing RS(2) to the diet of ileostomy subjects did not interfere with small-bowel absorption of nutrients or total sterols, except for a small increase in iron excretion. The ileostomy model seems to give reliable results for in vivo measurement of RS.

Predominance of caecal injury in a new dextran sulphate sodium treatment in rats: histopathological and fermentative characteristics.

OBJECTIVES: Cyclic administrations of dextran sulphate sodium (DSS) alternating with distilled water usually induce chronic colitis after a few weeks. In order to obtain stable chronic colitis (without recovery or relapse) in a few days, a new continuous DSS treatment was tested and characterized. Short-chain fatty acids (SCFAs), which remain poorly documented in experimental colitis, were also investigated. METHODS: Thirty-six Sprague-Dawley rats were treated with 5% DSS for 7 days (DI) followed by 3% DSS for 7 days (DM) or 14 days (DF). Control rats received only water. Inflammatory injuries in the caecum and the colon were assessed by macroscopic (colon length, caecum weight, damages score) and histological parameters. SCFAs (acetate, propionate, butyrate) were quantified individually in caecal, proximal and distal contents. RESULTS: Macroscopic and histological observations revealed that this continuous DSS treatment induced acute inflammation (DI) followed rapidly by chronic active colitis. The latter was uncommonly predominant in the caecum and the distal colon, and was also associated with some fermentative disturbances. Caecal SCFA concentrations decreased with DSS at DI and DM. The molar ratio of caecal butyrate increased with DSS. Acetate decreased in the colon while propionate increased. CONCLUSION: This new DSS treatment is able to induce in a few days stable chronic inflammation with caecal and distal predominant injuries, and mild fermentative caeco-colonic alterations. This model could contribute to the study of potential anti-inflammatory effects of prebiotics.

Advances in dietary fibre characterisation. 1. Definition of dietary fibre, physiological relevance, health benefits and analytical aspects.

Since 1953, the definition of 'dietary fibre' (DF) has evolved significantly following an international debate based on analytical progress, new nutritional and physiological knowledge and also private interests of the food industry. The overall tendency is towards an extension of the definition by including resistant starches as well as non-digestible oligosaccharides. This broadened definition is indeed based on physiological considerations as these compounds are not digested in the small intestine and become substrates for the colonic microflora, resulting in fermentation products that have a variety of local and possibly also systemic effects. A main reluctance to use this definition, however, is linked to the difficulty to quantify, with a universal method, the various compounds that fulfil the characteristics defined by this broad definition. At this point, if such a definition were adopted, there are two options, not necessarily antagonistic, which would be (1) to sum the content of NSP, resistant starches and non-digestible oligosaccharides quantified by distinct methods or (2) to use the Association of Official Analytical Chemists (AOAC) method of DF analysis (AOAC 985.29, 991.43) with complementary analyses of the different non-digestible oligosaccharides likely to be present in the food. With none of these solutions being fully satisfying from a scientific but also from a practical point of view, an innovative method has to be proposed within the next decade. The present review describes the various types of DF, effects of DF consumption on physiology and metabolism, past and current definitions, analytical aspects to measure DF and some aspects of DF claims and food labelling.

Advances in dietary fibre characterisation. 2. Consumption, chemistry, physiology and measurement of resistant starch; implications for health and food labelling.

Resistant starch (RS) is defined as 'the sum of starch and products of starch degradation not absorbed in the small intestine of healthy individuals'. This basic definition includes different types of starches that (1) are physically inaccessible, usually due to an encapsulation in intact cell walls, or (2) are naturally highly resistant to mammalian alpha-amylase, or (3) have been modified by hydrothermic treatments then retrograded. Interest in RS has increased significantly during the last two decades, mostly due to its capacity to produce a large amount of butyrate all along the colon. Butyrate has been observed to have a range of effects on cell metabolism, differentiation and cell growth as well as inhibition of a variety of factors that underlie the initiation, progression and growth of colon tumours. The physiological definition of RS, which seems to be nearly consensual, raises a difficulty in proper analytical quantification of RS. A number of methods have, however, been proposed and provide similar values for the RS content in most of the starch types and starchy foods. It seems, however, that some starches, proven to be partly resistant according to in vivo investigations on ileostomy subjects, could not be quantified by most of these methods. This may be due to a widespread use of glucoamylase during the first steps of these methods. Accordingly, there is an international debate on health aspects of RS and on how to quantify the RS content of food products. The present review describes aspects of classification of RS, past and current consumption, physiological effects and analytical aspects, and concludes with impacts on food and product labelling.

Physiological aspects of resistant starch and in vivo measurements.

Resistant starch (RS) is the sum of starch and products of starch degradation not absorbed in the small intestine of healthy individuals. There are a number of RS with different characteristics which may have a different fate in the colon. As a consequence, all RS should not be considered equivalent as far as physiological properties are concerned; indeed, they may have a different impact on colonic health. This statement may explain part of the apparent contradictions in the literature on RS and cancer or inflammatory disease prevention. RS is fermented in the large intestine into short-chain fatty acids and, among those, butyrate, which is recognized as the main nutrient of the colonocyte. This fermentation pattern seems to be responsible for most of the effects of RS on colonic health. Another important property is linked to its ability to lower colonic pH, which is usually considered as beneficial for mineral biovailability in the colon or cancer prevention. Due to their fate in the digestive tract, RS materials do not seem to have any significant impact on glucose absorption or metabolism. On the contrary, they may have a hypocholesterolemic effect, but available information is contradictory.

Maternal supplementation with n-3 long chain polyunsaturated fatty acids during perinatal period alleviates the metabolic syndrome disturbances in adult hamster pups fed a high-fat diet after weaning.

Perinatal nutrition is thought to affect the long-term risk of the adult to develop metabolic syndrome. We hypothesized that maternal supplementation with eicosapentaenoic acid and docosahexaenoic acid during pregnancy and lactation would protect offspring fed a high-fat diet from developing metabolic disturbances. Thus, two groups of female hamsters were fed a low-fat control diet, either alone (LC) or enriched with n-3 long chain polyunsaturated fatty acids (LC-PUFA) (LO), through the gestational and lactation periods. After weaning, male pups were randomized to separate groups that received either a control low-fat diet (LC) or a high-fat diet (HC) for 16 weeks. Four groups of pups were defined (LC-LC, LC-HC, LO-LC and LO-HC), based on the combinations of maternal and weaned diets. Maternal n-3 LC-PUFA supplementation was associated with reduced levels of basal plasma glucose, hepatic triglycerides secretion and postprandial lipemia in the LO-HC group compared to the LC-HC group. Respiratory parameters were not affected by maternal supplementation. In contrast, n-3 LC-PUFA supplementation significantly enhanced the activities of citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase compared to the offspring of unsupplemented mothers. Sterol regulatory element binding protein-1c, diacylglycerol O-acyltransferase 2, fatty acid synthase, stearoyl CoA desaturase 1 and tumor necrosis factor α expression levels were not affected by n-3 LC-PUFA supplementation. These results provide evidence for a beneficial effect of n-3 LC-PUFA maternal supplementation in hamsters on the subsequent risk of metabolic syndrome. Underlying mechanisms may include improved lipid metabolism and activation of the mitochondrial oxidative pathway.

Perinatal and postweaning exposure to galactooligosaccharides/inulin prebiotics induced biomarkers linked to tolerance mechanism in a mouse model of strong allergic sensitization.

Food allergies are increasing, and no treatment exists, thus enhancing interest in prebiotic strategies. This study aimed to analyze the preventive effects of prebiotic feeding during perinatal and postweaning periods in a mouse model of allergy by studying biomarkers related to tolerance (IgG2a, IgA, IFN-γ, TGF-ß, and IL-10), to allergy (IgE, IgG1, IL-4, IL-17, symptoms), and to microbiota (propionate and MyD88). Balb/c mice, both dams and their pups, were fed a diet supplemented with (+Prb) or without (-Prb) GOS/inulin prebiotics. Mice were then sensitized with allergens. Regardless of diet, sensitized mice exhibited similar levels of IgE, IgG1, CD-23, IL-4, IL-17, and symptoms. However, in comparison to -Prb-sensitized mice, +Prb-sensitized mice displayed higher concentrations of total IgG2a (6669 ± 1788 vs 3696 ± 1326 fluorescence units, p < 0.005), specific IgA (285 ± 26 vs 156 ± 9 fluorescence units, p < 0.01), IFN-γ (3194 ± 424 vs 1853 ± 434 pg/mL, p < 0.01), IL-10 (777 ± 87 vs 95 ± 136 pg/mL, p < 0.005), TGF-ß (4853 ± 1959 vs 243 ± 444 pg/mL, p < 0.01), MyD88 (0.033 ± 0.019 vs 0.009 ± 0.004 relative expression, p < 0.01), and propionate (4.15 ± 0.8 vs 2.9 ± 1.15 µmol, p < 0.05). In a mouse model of allergy, prebiotic exposure during perinatal and postweaning periods induced the highest expression of biomarkers related to tolerance without affecting biomarkers related to allergy.

Exposure to a galactooligosaccharides/inulin prebiotic mix at different developmental time points differentially modulates immune responses in mice.

Prebiotics constitute emerging tools to alleviate immune pathologies. This study aimed to evaluate the effect of prebiotic exposure during perinatal and postweaning periods on immune and gut regulations. Mice were fed either a galactooligosaccharides/inulin prebiotic mix-enriched diet or a control diet during the perinatal and/or postweaning periods. Biomarkers related to gut barrier function (SCFA, heat shock proteins, zonula occludens protein-1, and mucin-2) and immune mechanisms (IgA, IgE, IgG1, IgG2a, IL-10, TGF-ß, IL-4, IL-17A, and IFN-γ) were analyzed. The milk of dams fed the prebiotic diet was more concentrated in both IgA and TGF-ß when prebiotics were introduced during both the perinatal and postweaning periods; IL-10, IgA, and IgG2a were increased in pups; and expression of intestinal markers was more pronounced. Postweaning exposure to prebiotics alone induced higher INF-γ and TGF-ß levels, whereas IgA levels fell. Combined exposure periods (perinatal/postweaning) to prebiotics increased tolerance-related immunoglobulins in pups and reinforced gut barrier functions.

Long term metabolic impact of high protein neonatal feeding: a preliminary study in male rat pups born with a low birth weight.

BACKGROUND & AIMS: Nutrition received in early life may impact adult health. The aim of the study was to determine whether high protein feeding in neonatal period would have long term metabolic effects in an animal model of low birth weight infants. METHODS: Male rat pups born from dams receiving a low protein diet during gestation were separated from their mothers, and equipped with gastrostomy tubes to receive as their sole feeding a milk formula of either adequate protein (AP; n = 14; 8.7 g protein/dL; total energy: 155 kcal/100 g), or high protein content (HP; n = 14; 13.0 g protein/dL; total energy: 171 kcal/100 g) between the 7th (D7) and 21st day (D21) of life. Rats were then weaned to standard chow until sacrificed at adulthood. RESULTS: At D18, HP feeding was associated with higher estimated rates of protein turnover (p = 0.007) and synthesis (p = 0.051), as assessed using l-[U-(13)C]valine infusion. HP milk feeding in early life was associated with an increase in weight gain from puberty through adulthood, along with an increase in food intake, serum insulin (179 ± 58 vs. 55 ± 7 pmol/L; means ± SE), pancreatic ß-cell number, plasma triglycerides (95 ± 8 vs. 73 ± 9 mg/dL), serum leptin (9.7 ± 1.0 vs. 5.5 ± 1.2 ng/mL), mesenteric fat mass, and adipocyte size. CONCLUSIONS: In an animal model of low birth weight infants, high protein neonatal feeding may have a lasting effect on fat and glucose metabolism, potentially leading to "metabolic syndrome" in adulthood.

The definition of dietary fiber - discussions at the Ninth Vahouny Fiber Symposium: building scientific agreement.

A definition for dietary fiber was adopted in June 2009 by the Codex Alimentarius Commission based on the recommendation for endorsement of the Codex Committee on Nutrition and Foods for Special Dietary Uses (CCNFSDU) in November 2008. The definition listed three categories of carbohydrate polymers that are not hydrolyzed by the endogenous enzymes in the small intestine of humans. However, the definition left the inclusion of carbohydrates with degrees of polymerization (DP) in the range of 3 and 9 to the discretion of national authorities and left the 'physiological effect(s) of benefit to health' as undefined. The ILSI Europe and ILSI North America's committees on dietary carbohydrates organized a forum at the Ninth Vahouny Fiber Symposium in 2010 to discuss these implementation issues with the objective of building scientific consensus on how to resolve them. The results of this session are encouraging and indicated that the scientific community agrees on maintaining a worldwide consensus regarding the inclusion of non-digestible carbohydrates with ≥DP3 as dietary fiber and on a core, non-exhaustive list of beneficial physiological effects that dietary fibers have. These results are consistent with previous worldwide agreements.

Supplementation with galactooligosaccharides and inulin increases bacterial translocation in artificially reared newborn rats.

Supplementation of formulas with prebiotics enhances the growth of lactate producing bacteria, and fecal lactate, and acetate levels in infants. High concentrations of organic acids in intestinal lumen have, however, been shown to impair the intestinal barrier function. To determine whether stimulating the colonic microbiotal metabolism with prebiotics would impair the neonatal intestinal barrier function, artificially reared rats were fed milk formula with or without a mixture of galactooligosaccharides/inulin (GOS/Inulin, 88/12; 5.6 g/L) from the 7th d of life (d7) until weaning (d20). At d18, GOS/inulin supplementation had increased the concentrations of acetate and lactate in colonic lumen. Although neither ileum-associated microbiota nor colonic permeability (assessed in Ussing chambers), nor the expression of tight junction claudin-2 and claudin-3 mRNA were altered, GOS/inulin supplementation was associated with increased bacterial translocation (BT) toward spleen. None of these effects persisted at d40. We conclude that GOS/inulin supplementation may increase BT in an immature gut. The balance between the potential infectious risk of BT vs. its putative beneficial effect on the maturation of neonatal immune system clearly warrants further study.

Recovery of exfoliated cells from the gastrointestinal tract of premature infants: a new tool to perform "noninvasive biopsies?".

To gain insight into specific gene expression in the gastrointestinal (GI) tract of preterm infants, we adapted a method to isolate exfoliated epithelial cells. Gastric residual fluid aspirates (n = 89) or stool samples (n = 10) were collected from 96 neonates (gestational age, 24-36 wk). Cells were characterized by microscopic observation, cytokeratin-18 immunodetection, and expression of transcripts. The human origin of cellular DNA was confirmed by amplification of specific X and Y chromosome sequences. Isolation yielded 100-500 cells per sample for gastric aspirates (n = 8) and 10-20 cells for fecal samples (n = 5). Epithelial origin was confirmed by immunodetection of cytokeratin 18. Analyses of reverse transcribed products, using two independent methods, from 15 gastric fluid and two stool samples showed that 18S-rRNA and transcripts of beta-actin, glyceraldehyde-3-phosphate dehydrogenase (gapdh), and period1 were in quantities corresponding to at least 10 cells. On 59 aspirates, we found beta-actin transcripts (all but one), cytokeratin 18 (eight positive of eight samples), SLC26-A7-1 (13 positive of 19 samples), period2 (17 positive of 17 samples), and clock (25 positive of 26 samples). Exfoliated cells can be recovered from gastric aspirates and fecal samples and serve as a tool to investigate the impact of therapeutic and nutritional regimens on the maturation of GI functions.

Solid-phase microextraction method for carbon isotopic analysis of volatile carboxylic acids in human plasma by gas chromatography/combustion/isotope ratio mass spectrometry.

A new analytical method is described for the determination of the physiological concentration and low-level enrichment of (13)C-short-chain volatile organic acids (SCVAs) (e.g. (13)C-acetate and (13)C-butyrate) in human plasma. This two-step method involves solid-phase microextraction (SPME) coupled to gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) without any organic solvents or derivatizing agents. Two SCVA extraction methods were compared using a carboxen/polydimethylsiloxane fiber: headspace sampling (HS) and liquid sampling (LS) SPME. The influences of extraction temperature and time were tested to optimize the adsorption of SCVAs onto the fiber. The comparison of the peak area responses of the acids in the two adsorption methods showed better sensitivity in the human physiological concentration range in the LS mode than in the HS mode. The accuracy of isotopic enrichment measurement was determined using plasma spiked with (13)C-acetate and (13)C-butyrate solution from 0 to 1 mol percent excess (MPE). The linearity and repeatability (RSD < 5%) were measured in LS mode. Plasma SCVA concentrations were also determined relative to 3-methylvalerate (internal standard). Linearity and repeatability were observed from 0 to 400 microM for acetate, from 0 to 20 microM for propionate, and from 0 to 10 microM for butyrate. This method was also used to determine plasma acetate production obtained from lactulose (an undigestible disaccharide) fermentation in one healthy volunteer over 3 h. The acetate concentration increased twofold, 2 h after oral lactulose intake. These results are in agreement with the data obtained by GC/MS in healthy volunteers and obese adults following a lactulose intake by using higher amounts of labelled tracers.SPME coupled with GC/C/IRMS can be used to analyze (13)C-SCVAs at low enrichment (<0.5 MPE) within the physiological concentration measured in human plasma.

Cellular and biochemical features of skeletal muscle in obese Yucatan minipigs.

OBJECTIVE: To examine cellular and biochemical features of skeletal muscle in response to dietary-induced obesity in a novel Yucatan minipig model of childhood obesity. RESEARCH METHODS AND PROCEDURES: From 4 to 16 months of age, minipigs were fed either a recommended human-type diet (NF; n = 4) or were overfed a western-type diet with saturated fat and high-glycemic index carbohydrates (OF, n = 4). Muscle samples (biceps femoris) were histochemically stained for the identification of intramuscular adipocytes, intramyocellular lipid aggregates (oil red O), and myofiber types (myosin ATPase, succinate dehydrogenase). Gene expressions and/or activities of factors involved in lipogenesis, lipolysis, or energetic metabolism were quantified in muscle. RESULTS: Cross-sectional areas of myofibers paralleled pig body weight (r = 0.86, p < 0.01). The size of intramuscular adipocytes, the relative proportion of oil red O-stained fibers, and total muscle lipid content tended (p < or = 0.10) to increase in response to OF diet. Hormone-sensitive lipase, carnitine palmityl transferase-I, and uncoupling protein 2 mRNA levels were lower (p < 0.05) in OF pigs than in NF pigs. Activities of beta-hydroxyacyl-coenzyme A dehydrogenase and citrate synthase assessing post-carnitine palmityl transferase I events and the proportion of oxidative myofibers were not altered by OF diet. Activity and gene expression of fatty acid synthase were lower (p < 0.02) in OF pigs than in NF pigs. DISCUSSION: Overfeeding in Yucatan minipigs reduced the expression levels of three catabolic steps in skeletal muscle that are involved also in the etiology of human obesity.