Clinical and anthropometric measurements
Body weight, blood pressure, and waist circumference were recorded every 2 weeks. Body weight was measured by using a scale model Detecto Physician Beam Scale (HOSPEQ, Inc., Miami, FL). Waist circumference was measured by the same person according to the guidelines of the National Heart, Lung, and Blood Institute (NIH publication no. 00-4084). Blood pressure was measured with the use of an automatic blood pressure monitor (Omron Healthcare, Milton Keynes, UK).
Blood samples
OGTT was performed for both BF and D groups at baseline and at the end of the study (week 12) with a 75-g oral glucose challenge. Fasting blood samples were collected in the morning after a 12-h fast. Two-hour blood samples were taken at 30, 60, 90, and 120 min for the measurement of serum glucose and insulin. Serum was separated by centrifugation for 15 min at 1465 × g (3200 rpm) at 4○C and stored at −80°C until further analysis.
Breakfast, lunch, and dinner meal challenge
On the day of meal challenge, each subject is reported to the laboratory at 07:00 after an overnight fast. At 07:30, a catheter was placed in the antecubital vein of the nondominant arm and remained in the patient until 20:00. Fasting baseline blood sample was taken for measurement of glucose, insulin, and ghrelin. Each group consumed their assigned meal plan (Table 1), breakfast at 8:00, lunch at 13:00, and dinner at 19:00. The test meals were consumed in their entirety within 15 min. Venous blood samples were collected 30, 60, 120, and 180 min after breakfast, lunch, and dinner for glucose, insulin, and ghrelin. The appetite visual analogue scale scores (hunger and satiety) were concomitantly completed.
Appetite questionnaires
Appetite scores for hunger and satiety were assessed using 100-mm visual analogue scales [29] before and 30, 60, 120, and 180 min after breakfast, lunch, and dinner. Subjects were asked to make a single vertical mark on each scale somewhere between the 0 and 100 mm extremes (i.e., not at all hungry to very hungry) to indicate their feelings at that time-point.
Biochemical blood analyses
Serum glucose was determined by the glucose oxidase method (Beckman Glucose Analyzer, Fullerton, CA). Total and HDL cholesterol and triacylglycerols were measured enzymatically using a Hitachi-Cobas Bio centrifugal analyzer (Roche Diagnostics, Indianapolis, IN) using standard enzymatic kits (Roche Diagnostics). Low-density lipoprotein cholesterol (LDL-C) concentrations were calculated using the Friedewald equation as was described earlier [30]. Serum insulin was determined by a double antibody radioimmunoassay (CIS Bio International, Gif-Sur Yvette-Cedex, France). Plasma ghrelin was measured with an enzyme immunoassay kit (Phoenix Pharmaceuticals, Belmont, CA). Homeostasis model assessment indices of insulin resistance (HOMA-IR) and beta-cell (HOMA-B) function were calculated using the following formulas: HOMA-IR = fasting serum insulin (μIU/ml) × fasting serum glucose (mmol/l)/22.5; HOMA-B = 20 × fasting serum insulin (μIU/ml) / fasting glucose (mmol/l)-3.5 [31]. Insulin sensitivity index (ISI) was calculated using the following formula: ISI = 10,000/√((fasting glucose (mg/dl) × fasting insulin (μIU/ml)) × (mean glucose (mg/dl) × mean insulin (μIU/ml))) [32].
