Subjects
Ten healthy men [age 24 ± 0.6 years, BMI 22 ± 0.2 kg/m2 (mean ± SEM)] were recruited. The main criteria for exclusion were use of medication, blood donation, or use of antibiotics in the past three months, gastrointestinal surgery or dysfunction, inflammatory diseases, and diabetes mellitus. Approval was obtained from the Medical Ethics Committee of the ‘Beoordeling Ethiek Biomedisch Onderzoek’ foundation, Assen, The Netherlands. Each subject gave written informed consent for the study. This trial was registered at trialregister.nl as NTR3020.
Experimental design
In the overall study design, addressing two different research questions, four different wheat-based products were tested. To increase clarity and be able to focus on the results of one of these questions, only two meals are described in this paper. A description of the other products and the results can be found elsewhere [19]. The study was performed in a randomized, crossover manner, with at least 1 week between each study day. The subjects were asked to refrain from consuming foods naturally high in 13C, like cane sugar, corn products, and pineapple, for 3 days preceding the experiments and from alcohol consumption and strenuous exercise for 24 h before each study day. Food intake on the day before each study day was individually standardized. To minimize variation, a standard evening meal was provided at the commercial research facility (QPS Netherlands B.V.), where the participants stayed overnight. In the evening, a venous catheter was inserted in each forearm for blood collection and for infusion of the tracer D-[6,6-2H2]glucose (98 % 2H atom percent excess, Isotec) [20, 21]. Subjects fasted overnight, but were allowed to drink water. In the morning (t = −122 min), a bolus of 26.7 mL D-[6,6-2H2]glucose solution (80 × 0.07 mg/kg body weight) was injected within 2 min, and a continuous infusion of 0.07 mg/kg body weight D-[6,6-2H2]glucose per min was started (t = −120 min) and maintained for 8 h (until t = 360 min). The bolus amount was 80× the infusion rate over 1 min, according to the paper of Tissot et al [20]. The infusion rate over 1 min was 0.07 mg/kg BW [21], to reach a good steady state. So the continuous infusion contained 4.2 mg/kg BW per 60 min, administered at a desired infusion rate of 20 mL/h, resulting in the concentration of 0.21 mg/mL/kg BW. The bolus had the same concentration, and therefore, to administer 80 × 0.07 mg/kg BW an amount of 26.7 mL was administered.
Two hours after the start of the infusion the test meal was ingested (t = 0 min). Figure 1 shows a simplified time line of each study day. Water (150 mL) was provided hourly, starting at t = 120 min. During the study period physical activity was limited.
Test meals
Two types of wheat bread were prepared (TNO, Zeist, The Netherlands), with the same composition, but a different structure, due to different milling and bread making processes; a kernel bread (KB) prepared with flour and 85 % broken wheat kernels, and a control bread (CB) made from wheat flour, combined with wheat bran, to obtain the same dietary fiber content as in the KB.
To provide the necessary ingredients, unlabeled (1.085 at.% 13C) wheat grains [T. aestivum var Capo, grown in Austria] were milled in different ways; by conventional roller-milling the wheat kernels were milled to obtain refined white flour and wheat bran, and broken wheat kernels were obtained using a breaker mill. For 13C-enrichment of the products, 13C-labeled wheat [T. aestivum var Paragon (1.359 at.% 13C)], cultured in a 13CO2 enriched atmosphere, was used. A small part of the 13C-labeled wheat kernels was milled to obtain a fine wholemeal wheat flour, and the other part was broken to obtain broken kernels that were similar to the unlabeled broken kernels.
CB was prepared with 1446 g unlabeled white wheat flour, 240 g 13C-labeled wholemeal wheat flour, 314 g wheat bran, 1300 g water, 33.4 g yeast, 36 g salt, 3 g malt, and 70 ppm ascorbic acid. After kneading, the dough was left to rise for 30 min, molded, and left to rise for 60 min. KB was prepared with 264 g unlabeled white wheat flour, 36 g 13C-labeled wholemeal wheat flour, 1496 g unlabeled broken wheat kernels, and 204 g 13C-labeled broken wheat kernels. All broken kernels were soaked in 1000 g water overnight at 10 °C. The following day, the soaked kernels were mixed with the flour, 230 g water, 33.4 g yeast, 36 g salt, 3 g malt, and 70 ppm ascorbic acid. The resulting dough was left to rise for 30 min, molded, and left to rise for 50 min. Subsequently, the breads were baked for 30 min at 240 °C. Bread slices were stored at −20 °C until use.
All test meals provided 50 g available carbohydrates; for the CB, the portion size was 138 g and for the KB 137 g. The breads were consumed together with 10 g light margarine (4 g fat), 2 slices lean ham (5 g fat, 6 g protein), and 250 mL tap water within 20 min. We assume that the digestibility of starch from both test products is similarly affected by this addition.
Bread characterization
Starch, dietary fiber, and moisture content were determined at Eurofins Analytico Food, The Netherlands. To quantify starch fractions (GT, GRA, GSA, GTA, and RS) of the test meals in vitro, an adapted version of the Englyst method [22] was used [23].
Particle size distribution of the broken kernels was determined by sieve analysis in duplicate.
Bread products volume (mL) was determined in triplicate by rapeseed displacement (AACCI Method 10-05.01 [24]) and, together with the bread weight (g), the product overall specific volume (mL/g) and density (g/mL) were obtained.
Breads were further characterized using microscopy and X-ray microtomography (XRT, porosity). A detailed description of both techniques can be found in Online Resource 1.
Sample collection
Blood was collected into several blood collection tubes (BD Diagnostics): 2 mL fluoride tubes (NaF) for glucose and bile acid measurements, 3 mL EDTA tubes [+30 µL DPP-4 inhibitor (Millipore)] for GIP, GLP-1, glucagon, and insulin determination, and 3 mL lithium/heparin tubes (CCK). Three basal blood samples were collected (t = −60, −30, −15 min), and postprandial samples were drawn every 15 min for 2 h, every 30 min for an additional 3 h, and once after 6 h. To obtain heparin plasma, blood was collected at t = −60, t = −15 min and then every 30 min for the first 3 h, and hourly for the last 3 h. After centrifugation (1300×g for 10 min at 4 °C), plasma aliquots were stored at −20 °C (NaF and EDTA plasma) or −80 °C (heparin plasma) until analysis.
Breath samples were collected by breathing through a straw into 10 mL Exetainer® vials (Labco Limited). Two basal breath samples were collected (t = −30, t = −5 min), and after the test meal, a sample was taken every 30 min until t = 360 min.
To get an impression about the impact of the breads on appetite, subjects were asked to rate their feeling of appetite (hunger) using a visual analog scale (VAS) at 15 min before and hourly after the test meal, although the number of subjects (n = 10) is insufficient to detect differences in these subjective measures [25]. At the same time points, their feeling and extent of discomfort (abdominal pain, flatulence, other complaints) was recorded (0 = no complaints, 3 = severe complaints). Shortly after consumption of the meal, the subjects scored (VAS) how tasty the meal was.
Measurement of plasma concentrations
Plasma glucose concentrations were measured on a Roche/Hitachi Modular automatic analyzer (Roche Diagnostics, Hitachi) using a glucose hexokinase method. The within- and between-run CV were ≤2 %. The ARCHITECT® insulin assay (Abbott Laboratories) was used to determine insulin concentrations in plasma. The total CV of this chemiluminescent microparticle immunoassay was ≤7 %. The glucagon assay was directed against the C-terminal of the glucagon molecule (antibody code no. 4305) and therefore measures glucagon of mainly pancreatic origin [26]. Total GIP was measured using the C-terminally directed antiserum (no. 80867) [27], which reacts fully with intact GIP (1-42) and the N-terminally truncated metabolite GIP (3-42). Total GLP-1 concentrations were determined as previously described [28], using a radioimmunoassay (antiserum no. 89390) specific for the C-terminal of the GLP-1 molecule and reacting equally with intact GLP-1 and the primary (N-terminally truncated) metabolite. The glucagon and incretin assays have detection limits of <2 pmol/L, and an intra-assay coefficient of variation of approximately 6 %. CCK was measured using antiserum no. 92128, which binds the bioactive forms of CCK with equal potency without cross-reactivity with any gastrin [29]. The detection limit of the CCK assay is 0.1 pmol/L, and the intra-assay coefficient of variation approximately 5 %.
Measurement of plasma bile acids
Fasting and postprandial concentrations of 15 individual BAs were determined using LC/MS: the primary BAs [cholic acid (CA) and chenodeoxycholic acid (CDCA)], the secondary BAs [deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA)], as well as their glycine (G, glyco-) and taurine (T, tauro-) conjugates. The lower limit of quantitation (LOQ) was 0.05 µM. Concentrations of TCA, LCA, GLCA, TLCA, TDCA, UDCA, and TUDCA were below the LOQ. The intra- and inter-assay CV ranged from 1.6 to 11.3 and 4.4 to 13.3 %, respectively. For sample preparation, 250 µl of internal standard solution was mixed with 25 µl plasma and centrifuged at 15.900×g for 10 min. The supernatant was transferred into a new vial, evaporated under nitrogen at 40 °C, and reconstituted in 100 µl of 50 % methanol. The solution was filtered with a 0.2-µm centrifugal filter at 2000×g for 10 min. After this step, the samples (injection volume 10 µl) were ready for analysis, using two different LC/MS systems. A detailed description of the systems and settings can be found in Online Resource 2.
Analysis of isotopic enrichment (breath and plasma) and calculations
Analysis of 13C abundance in breath CO2 was performed using GC/IRMS (Delta Plus XL; Thermo Fisher Scientific) measuring the 13C/12C ratio versus the international standard Pee Dee Belemnite (δ13CPDB, in ‰).
Plasma sample preparation required for analysis of isotopic enrichment by GC (derivatization) is described in detail elsewhere [30, 31]. 2H enrichment was measured by GC/MS as previously described [30], and 13C/12C isotope ratio was measured using GC/C/IRMS as previously described [32], both with some modifications [33].
Calculation of glucose kinetics was performed as previously reported [34].
Incremental areas under the curve (iAUC)
To determine differences in glucose kinetics and plasma glucose, insulin, glucagon, incretin, CCK, and bile acid concentrations, the 0–2 and 0–6 h iAUCs were calculated as previously described [34].
Statistics
Human data are presented as mean ± SEM, n = 10. Baseline-subtracted values are shown in the figures and were used in the analyses as well, for clarity, consistency, and comparison of variables. The overall study involved testing of four test meals. From the statistical analysis of the full experiment, we highlight only the comparisons involving the products of current interest. For parameters that result in individual points of a time curve, we fitted a model that accounts for the differences between the 10 subjects and the four occasions, the main effects of treatment and time, respectively, and the time × treatment interaction. Residual plots based on such a model were used to assess approximate normality and homogeneity of variances. Parameters that were not compatible with these assumptions were transformed before definitive analysis. Most variables were log-transformed, except for glucose (reciprocal values), VAS scores (angular transformation), RaE, and 13CO2 values (Poisson regression model on log scale).
We performed F tests on time × treatment interaction effects to assess whether the four test meals gave rise to curves of a different shape. If the test resulted in a P value <0.05, we tested differences between the meals for each time point. If there were no statistically significant differences in shapes, we tested overall differences between the meals based on the above model without the time × treatment interaction.
To assess summary measures such as iAUC, we fitted a model accounting for the differences in the means of the 10 subjects and the four occasions, and the main effects of treatment. Transformed data were used whenever residual plots showed incompatibility with normality and homogeneity assumptions. We performed an F test to see whether there were differences between the meals. If this test resulted in a P value <0.05, pairwise comparisons among the meals were conducted.
A Benjamini–Hochberg correction was applied on P values from all partial tests to correct for multiple comparisons, resulting in a set of differences in which at most 5 % were falsely selected (P < 0.0052 for partial tests was considered significant). The analyses were performed with the software package GenStat, release 13. The within-subject relationship (correlation) between variables was tested by regression analysis according to the method of Bland and Altman [35]. Test meal differences (e.g., density, porosity) were assessed using a Student’s t test; a P value <0.05 was considered significant. These analyses were performed using SPSS 20.0 for Windows (SPSS Inc., Chicago).