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Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan–microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health. Key points It is important to understand how carbohydrates are digested—both by human enzymes and by microorganisms present in the gut. This protocol is designed to characterize and quantify food and fecal polysaccharides at the monosaccharide, linkage and polysaccharide level. Analysis is performed by LC-MS/MS. Higher-throughput sample preparation in 96-well plates is possible by using a custom-made clamp to hold the plate lids closed during heating. Complex carbohydrates that are not broken down by human enzymes are food sources for gut microbiota. Toward understanding this process, this protocol describes the quantitative analysis of carbohydrates in food and fecal samples by using LC-MS/MS.

Background and Objectives: To determine the effect of a low carbohydrate diet and standard carbohydrate counting on glycaemic control, glucose excursions and daily insulin use compared with standard carbohydrate counting in participants with type 1 diabetes. Methods and Study Design: Participants (n=10) with type 1 diabetes using a basal; bolus insulin regimen, who attended a secondary care clinic, were randomly allocated (1:1) to either a standard carbohydrate counting course or the same course with added information on following a carbohydrate restricted diet (75 g per day). Participants attended visits at baseline and 12 weeks for measurements of weight, height, blood pressure, HbA1c, lipid profile and creatinine. They also completed a 3-day food diary and had 3 days of continuous subcutaneous glucose monitoring. Results: The carbohydrate restricted group had significant reductions in HbA1c (63 to 55 mmol/mol (8.9-8.2%), p<0.05) and daily insulin use (64.4 to 44.2 units/day, p<0.05) and non-significant reductions in body weight (83.2 to 78.0 kg). There were no changes in blood pressure, creatinine or lipid profile and all outcomes in the carbohydrate counting group were unchanged. There was no change in glycaemic variability as measured by the mean amplitude of glycaemic excursion in either group. Conclusions: A low carbohydrate diet is a feasible option for people with type 1 diabetes, and may be of benefit in reducing insulin doses and improving glycaemic control, particularly for those wishing to lose weight.

Background/objectivesConsumption of whole vs. refined grain foods is recommended by nutrition or dietary guideline authorities of many countries, yet specific aspects of whole grains leading to health benefits are not well understood. Gastric emptying rate is an important consideration, as it is tied to nutrient delivery rate and influences glycemic response. Our objective was to explore two aspects of cooked rice related to gastric emptying, (1) whole grain brown vs. white rice and (2) potential effect of elevated levels of slowly digestible starch (SDS) and resistant starch (RS) from high-amylose rice.Subjects/methodsTen healthy adult participants were recruited for a crossover design study involving acute feeding and testing of 6 rice samples (50 g available carbohydrate). Gastric emptying rate was measured using a 13C-labeled octanoic acid breath test. A rice variety (Cocodrie) with high-amylose content was temperature-cycled to increase SDS and RS fractions.ResultsIn vitro starch digestibility results showed incremental increase in RS in Cocodrie after two temperature cycles. For low-amylose varieties, SDS was higher in the brown rice form. In human subjects, low-amylose and high-amylose brown rice delayed gastric emptying compared to white rices regardless of amylose content or temperature-cycling (p < 0.05).ConclusionsWhole grain brown rice had slower gastric emptying rate, which appears to be related to the physical presence of the bran layer. Extended gastric emptying of brown rice explains in part comparably low glycemic response observed for brown rice.

OBJECTIVE: To investigate the effect of flexible intensive insulin therapy (FIIT) and an automated bolus calculator (ABC) in a Danish type 1 diabetes population treated with multiple daily injections. Furthermore, to test the feasibility of teaching FIIT in a 3-h structured course. RESEARCH DESIGN AND METHODS: The BolusCal Study was a 16-week randomized, controlled, open-label, three-arm parallel, clinical study of 51 adults with type 1 diabetes. Patients aged 18–65 years in poor metabolic control (HbA1c 8.0–10.5%) were randomized to the Control (n = 8), CarbCount (n = 21), or CarbCountABC (n = 22) arm. During a 3-h group teaching, the Control arm received FIIT education excluding carbohydrate counting. CarbCount patients were taught FIIT and how to count carbohydrates. CarbCountABC group teaching included FIIT and carbohydrate counting and patients were provided with an ABC. RESULTS: At 16 weeks, the within-group change in HbA1c was –0.1% (95% CI –1.0 to 0.7%; P = 0.730) in the Control arm, –0.8% (–1.3 to –0.3%; P = 0.002) in the CarbCount arm, and –0.7% (–1.0 to –0.4%; P < 0.0001) in the CarbCountABC arm. The difference in change in HbA1c between CarbCount and CarbCountABC was insignificant. Adjusting for baseline HbA1c in a regression model, the relative change in HbA1c was –0.6% (–1.2 to 0.1%; P = 0.082) in CarbCount and –0.8% (–1.4 to –0.1%; P = 0.017) in CarbCountABC. Treatment satisfaction measured by the Diabetes Treatment Satisfaction Questionnaire (status version) improved in all study arms, but the improvement was significantly greater in CarbCountABC. CONCLUSIONS: FIIT and carbohydrate counting were successfully taught in 3 h and improved metabolic control and treatment satisfaction. Concurrent use of an ABC improved treatment satisfaction further.

Background The prevalence of obesity is increasing worldwide and prevention is needed. Whole grain has shown potential to lower the risk of obesity, cardiovascular disease and type 2 diabetes. One possible mechanism behind the benefits of whole grain is the gut fermentation of dietary fiber (DF), e.g. non-starch polysaccharides and resistant starch (RS), in whole grain. The purpose of the study is to investigate the effect of whole grain rye-based products on glucose- and appetite regulation. Method Twenty-one healthy subjects were provided four rye-based evening test meals in a crossover overnight study design. The test evening meals consisted of either whole grain rye flour bread (RFB) or a 1:1 ratio of whole grain rye flour and rye kernels bread (RFB/RKB), with or without added resistant starch (+RS). White wheat flour bread (WWB) was used as reference evening meal. Blood glucose, insulin, PYY, FFA, IL-6 as well as breath H 2 and subjective rating of appetite were measured the following morning at fasting and repeatedly up to 3.5 h after a standardized breakfast consisting of WWB. Ad libitum energy intake was determined at lunch, 14.5 h after evening test and reference meals, respectively. Results The evening meal with RFB/RKB + RS decreased postprandial glucose- and insulin responses (iAUC) ( P  < 0.05) and increased the gut hormone PYY in plasma the following morning 0–120 min after the standardized breakfast, compared to WWB ( P  = 0.01). Moreover, RFB increased subjective satiety and decreased desire to eat, and both RFB and RFB/RKB decreased feeling of hunger (AUC 0–210 min). All rye-based evening meals decreased or tended to decrease fasting FFA ( P  < 0.05, RFB/RKB: P  = 0.057) and increased breath hydrogen concentration (0–120 min, P  < 0.001). No effects were noted on energy intake at lunch or inflammatory marker IL-6 (0 + 180 min) after the rye-based evening meals, compared to WWB. Conclusion Whole grain rye bread has the potential to improve cardiometabolic variables in an 11–14.5 h perspective in healthy humans. The combination RFB/RKB + RS positively affected biomarkers of glucose- and appetite regulation in a semi-acute perspective. Meanwhile, RFB and RFB/RKB improved subjective appetite ratings. The effects probably emanate from gut fermentation events. Trial registration The study was registered at: ClinicalTrials.gov, register number NCT02347293 ( www.clinicaltrials.gov/ct2/show/NCT02347293 ). Registered 15 January 2015.

The addition of vegetable to carbohydrate-based meals was shown to contribute to glycaemic management. The aim of this study was to investigate the impact of homogenisation on vegetables added to rice meals in terms of acute glycaemic responses (GR). In a randomised crossover trial, sixteen healthy volunteers completed thirteen test sessions, which included two sessions for glucose control, two for rice and nine for different vegetable-rice mixed meals: cooked pak choi and cooked rice (CP+R); cooked cauliflower and cooked rice (CC+R); cooked eggplant and cooked rice (CE+R); and their homogenised counterparts, both raw or cooked. Postprandial GR tests, in vitro carbohydrate digestion and chemical analyses were carried out for each test meal. Compared with pure rice, CE+R, CP+R and CC+R meals achieved significantly lower glycaemic indexes (GI) of 67, 71 and 73, whereas their homogenised counterparts failed to show significant difference with rice. The hydrolysis indexes (HI) of CE+R, CP+R and CC+R were 69·6, 83·8 and 80·6 % of the HI of the rice control. CE had the greatest effect on lowering the GI, the incremental area under the blood glucose curve from 0 to 120 min, the peak glucose value, the maximum amplitude of glucose excursion in 0–120 min (MAGE0 –120), the HI and rapid available starch. Both in vitro and in vivo tests demonstrated that incorporating non-homogenised cooked vegetables into a rice meal could slow the carbohydrate digestion and improve postprandial GR. Texture properties of vegetable may play an important role in underlying glycaemic control mechanisms.

A novel strategy for the qualitative analysis of carbohydrates is developed, utilizing a direct catalytic fuel cell (DCFC) as a sensor, combined with chemometric tools for processing the resulting response curves. Specifically, carbohydrate solutions were incubated with yeast to produce alcohol, and the corresponding current decay trends were measured using a direct catalytic fuel cell designed for ethanol detection. Multiple data processing approaches were then evaluated. Initially, the entire set of data points from the response curves was analyzed using principal component analysis (PCA). To reduce analysis time, chemometric processing was subsequently restricted to the initial portion of the response curves. Finally, to enhance the results, the current decay curves were analyzed in conjunction with the linear fitting parameters derived from the quasi-linear region of the initial response curves, utilizing the common dimension (ComDim) algorithm.

Although the effects of dietary fat and carbohydrate on satiety are well documented, little is known about the impact of these macronutrients on food hedonics. We examined the effects of ad libitum and isoenergetic meals varying in fat and carbohydrate on satiety, energy intake and food hedonics. In all, sixty-five overweight and obese individuals (BMI=30·9 (sd 3·8) kg/m2) completed two separate test meal days in a randomised order in which they consumed high-fat/low-carbohydrate (HFLC) or low-fat/high-carbohydrate (LFHC) foods. Satiety was measured using subjective appetite ratings to calculate the satiety quotient. Satiation was assessed by intake at ad libitum meals. Hedonic measures of explicit liking (subjective ratings) and implicit wanting (speed of forced choice) for an array of HFLC and LFHC foods were also tested before and after isoenergetic HFLC and LFHC meals. The satiety quotient was greater after ad libitum and isoenergetic meals during the LFHC condition compared with the HFLC condition (P=0·006 and P=0·001, respectively), whereas ad libitum energy intake was lower in the LFHC condition (P<0·001). Importantly, the LFHC meal also reduced explicit liking (P<0·001) and implicit wanting (P=0·011) for HFLC foods compared with the isoenergetic HFLC meal, which failed to suppress the hedonic appeal of subsequent HFLC foods. Therefore, when coupled with increased satiety and lower energy intake, the greater suppression of hedonic appeal for high-fat food seen with LFHC foods provides a further mechanism for why these foods promote better short-term appetite control than HFLC foods.

Background Fortifying human milk contributes to the prevention of postnatal growth failure in preterm infants. Because of the natural variability of human milk, targeted fortification of human milk has been advocated. However, data regarding the efficacy and safety of prolonged targeted fortification are scarce. We aimed to assess the safety of targeted fortification of human milk in preterm infants compared with standard fortification, as well as the effects on infant growth. Methods We conducted an interventional study during hospital stay in healthy very low birth weight preterm infants who were exclusively fed human milk. Pools of human milk collected for 24 h were analysed using mid-infrared transmission spectroscopy. Targeted fortification of human milk was performed by adding macronutrients to native human milk to obtain optimal ratios of fat (4.4 g), carbohydrates (8.8 g), and protein (3 g) per 100 ml. The intervention period lasted 4–7 weeks. Weekly weight and daily growth rates were compared with those of a standardized fortification group of very low birth weight preterm infants who received standard fortified human milk (n = 10). The osmolality as well as the metabolic and gastrointestinal tolerance were monitored. Intergroup differences were evaluated using the Mann–Whitney U-test. Results A total of 10 preterm infants (birth weight 1223 ± 195 g; gestational age 29.1 ± 1.03 weeks) were enrolled and 118 samples of pooled milk were analysed. On average, 1.4 ± 0.1 g of protein, 2.3 ± 0.5 g of carbohydrate, and 0.3 ± 0.1 g of fat per 100 ml were added to the milk. Osmolality values after target fortification were within recommended limits (376 ± 66 mOsml/kg). Weekly weight gain (205.5 g; 95 % CI 177–233 vs 155 g; 95 % CI 132–178; p = 0.025) and daily growth rates (15.7 g/kg/day; 95 % CI 14.5–16.9 vs 12.3 g/kg/day; 95 % CI 10.7–13.9; p = 0.005) were higher in infants receiving target fortification than in infants receiving standardized fortification. The infants receiving targeted fortified milk consumed similar volumes as infants in the standardized fortification group (148 ± 4.5 vs 146 ± 4 ml/kg/day). No signs of either gastrointestinal or metabolic intolerance were observed. Conclusions Target fortification appears to promote growth in very low birth weight preterm infants without any detrimental effects. Trial registration NCT02716337

Dietary nutrients have significant effects on the risk of cardiovascular diseases. However, the results were not uniform across different countries. The study aims to determine the relative importance of dietary nutrients associated with coronary artery disease (CAD) among the Nepalese population. A hospital-based matched case-control study was carried out at Shahid Gangalal National Heart Center in Nepal. In the present study, patients with more than seventy percent stenosis in any main coronary artery branch in angiography were defined as cases, while those presenting normal coronary angiography or negative for stressed exercise test were considered controls. Dietary intakes of 612 respondents over the past 12 months were evaluated using a semi-quantitative customized food frequency questionnaire. In conditional regression model, the daily average dietary intake of β-carotene (OR: 0.54; 95%CI: 0.34, 0.87), and vitamin C (OR: 0.96; 95%CI: 0.93, 0.99) were inversely, whereas dietary carbohydrate (OR: 1.16; 95%CI: 1.1, 1.24), total fat/oil (OR: 1.47; 95%CI: 1.27, 1.69), saturated fatty acid (SFA) (OR: 1.2; 95%CI: 1.11, 1.3), cholesterol (OR: 1.01; 95%CI: 1.001, 1.014), and iron intakes (OR: 1.11; 95%CI: 1.03, 1.19) were positively linked with CAD. Moreover, in random forest analysis, the daily average dietary intakes of SFA, vitamin A, total fat/oil, β-carotene, and cholesterol were among the top five nutrients (out of 12 nutrients variables) of relative importance associated with CAD. The nutrients of relative importance imply a reasonable preventive measure in public health nutrients specific intervention to prevent CAD in a resource-poor country like Nepal. The findings are at best suggestive of a possible relationship between these nutrients and the development of CAD, but prospective cohort studies and randomized control trials will need to be performed in the Nepalese population.