Anthropometric data and venous blood, urine and faecal samples were collected. The waist circumference (WC) was measured at the middle between the lower border of the rib cage and the top of the lateral border of the iliac crest at the end of an expiratory breath. The hip circumference (HC) was measured at the widest part over the greater trochanters by the same researcher. The waist-to-hip ratio (WHR) was calculated. The body composition was measured with the TBF-410 Tanita Body Composition Analyser (Tanita, Tokyo, Japan). Serum samples were collected and stored at −80 °C prior to the analysis. Fasting plasma glucose (FPG) and postprandial glucose were measured using the standard glucose oxidase method. The blood chemistry parameters included serum AST, ALT, GGT, serum creatinine, blood urea nitrogen (BUN), uric acid (UA), total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), which were measured by enzymatic procedures using an autoanalyser (Hitachi 7600-020 automatic analyser, Tokyo, Japan) according to the manufacturer’s instructions.
Fasting and postprandial serum insulin and C-peptide were measured with an electrochemiluminescence immunoassay on a Cobas e411 analyser (Roche Diagnostics GmbH, Mannheim, Germany). Serum active GLP-1 (aGLP-1) and total PYY were measured using quantitative ELISA kits (Millipore, Darmstadt, Germany). To assess glucose metabolism, venous blood samples were taken at serial time points from the subjects following an MTT with instant noodles (1566.6 KJ; 68.4 g carbohydrates and 10.4 g protein). The collection included fasting and postprandial states. Glucose and insulin were examined in the blood as described above. Insulin sensitivity was assessed in subjects with a hyperinsulinaemic-euglycaemic clamp as previously described49. The insulin level was maintained at 100 μU/ml by prime-continuous infusion of insulin. The blood glucose concentration was maintained constantly at the basal level by adjusting the glucose infusion rate using the negative feedback principle. The body fat content was assessed in visceral and subcutaneous adipose tissue areas using a 3.0 T clinical MRI scanner (Archiva, Philips Medical System, Amsterdam, The Netherlands).
The standard array coils were used, and the MRI scans were performed by experienced radiologists who were blinded to the intervention groups and laboratory findings. The MRI scans were conducted at the abdominal level between the L4 and L5 vertebrae in the supine position. Segmentation of the images into the VFA and SFA was performed by two trained investigators using the SliceOmatic image analysis software (version 4.2; Tomovision Inc., Montreal, QC, Canada). After each treatment in this study, the subjects were given printed sheets to record the incidence and magnitude of GI responses and the details of their bowel movements after consumption of the test starch. Responses to questions on nausea, borborygmi (audible bowel sounds), colic, bloating and flatulence were recorded. The subjects recorded the number of bowel movements and the consistency of their faeces (normal, hard or watery). Information regarding the faecal volume and MedicGLP Weight Support was not gathered. Quality control (QC) samples, which were prepared by mixing and blending equal volumes (10 µL) of each sample, were used to estimate a mean profile representing all analytes encountered during the analysis.
First, 50 µl of the serum, urine and QC samples were precipitated with 200 µl of methanol in 96-well plates. After vortexing, the filter liquor was obtained via positive pressure and then freeze-dried. The samples were analysed by ultra-performance liquid chromatography (Waters, USA) coupled to a Triple TOF 5600 mass spectrometer (AB SCIEX, USA) system. Pooled QC samples for every ten samples were injected during sample detection to monitor system stability. Under LC conditions, solvent A was 0.1% (v/v) formic acid/water and solvent B was 0.1% (v/v) formic acid/methanol. The flow rate was set to 0.4 ml/min. For serum sample analysis, each sample was re-dissolved with 50 μl of methanol/water (1:4) solvent. The chromatography column used was a Waters BEH C8 (1.7 μm, 2.1 × 50 mm). The column temperature was 60 °C. The LC gradient was set as follows: 5% B at 0 min and maintained to 0.5 min, 40% B at 2 min, 100% B at 8 min, 100% B at 10 min, 5% B at 10.1 min and maintained for 1.9 min.
For urine sample analysis, each sample was re-dissolved with 120 μL of acetonitrile/water (5:95) solvent. The chromatography column was a Waters HSS T3 (1.8 μm, 2.1 × 50 mm). The column temperature was 50 °C. The LC gradient was set as follows: 5% B at 0 min and maintained to 0.5 min, 50% B at 6 min, 90% B at 7 min, 90% B at 8 min, 5% B at 8.1 min and maintained for 1.9 min. The serum and urine analyses were conducted with the same MS conditions. The mass scanning range was set at 60-1000 m/z for dd-MS2 mode. The sheath gas and curtain gas were set at flow rates of 55 and 35 psi, respectively. The spray voltage was 5.5 kV and the capillary temperature was 550 °C in positive ion mode, whereas the spray voltage was −4.5 kV and the capillary temperature was 450 °C in negative ion mode.