This study, including the experimental details, protocols and informed consent, received research funding and ethical approval from the Joint Committee of Ethics and Research of International Medical University (IMU): approval no. 4.18/JCM-84/2014.
Informed consent was obtained from all participants prior to screening. Fifty four eligible Malaysian subjects participated in this study. The majority were students and staff of IMU. Inclusion criteria were: ages from 18 to 30 years; and body mass indices (BMIs) between 18 and 27.4 Kg/m2, as specified for non-obese in the Malaysian Clinical Practice Guidelines, 2004. Exclusion criteria were: a history of diabetes; pregnancy; current smokers; and subjects with an illness (or using medication) known to affect salivary amylase secretion (including the use of orthodontic devices).
Salivary amylase estimation
A chromogenic kinetic reaction assay kit (1–1902; Salimetrics In, USA) was used to determine the activity of amylase in saliva samples. The enzymatic substrate in this kit was 2-chloro-p-nitrophenol coupled to maltotriose. Salivary amylase releases 2-chloro-p-nitrophenol from this substrate, which was then determined from its Abs405nm. Volumes of saliva, standards and reagents were all half the volumes recommended in the brochure supplied with the kit.
Each participant fasted overnight for a minimum of 8 h. To minimise circadian variability in the salivary secretory rates, saliva collection was undertaken between 7 a.m. and 9 a.m. on the following morning, After rinsing the mouth with water (to reduce contamination of the collected sample with solid particulates) each subject’s non-stimulated saliva sample was collected in a weighed polypropylene tube by the passive drool method  for exactly 2 min. Subsequently a stimulated saliva sample was collected for 2 min in a similar manner. The stimulus was 2 % (w/v) acetic acid, applied at the dorsum of the tongue, using a cotton tip . After reweighing tubes to determine the salivary flow rate, the samples were centrifuged at 2000 x g for 10 min at 4 °C, then stored in aliquots of 100 μL at −20 °C for future analysis.
For determination of amylase activities, samples were thawed completely, mixed, centrifuged at 3000 rpm for 15 min at 37 °C, and then diluted 200-fold in the diluent provided in the assay kit. Fixed volumes (4 μL) of controls, standards and diluted saliva samples were added in replicates of 3 to wells of a 96 well plate. 160 μL of the α-amylase substrate solution was added to each well and the plate was immediately placed in a microplate reader, which was maintained at 37 °C throughout the incubation. The reader was programmed to read Abs405nm at 1 min and at 3 min. The readings at 1 min were subtracted from the readings at 3 min. The resulting increases in absorbance provided a measure of the relative rate of salivary amylase activities. An in-house control saliva sample, also assayed in replicates of 3, was included in each assay, to normalize the results for any systematic inter-assay variability. The amylase activities of high and low activity standards with known amylase concentrations that were provided in the kit, were used to calibrate the enzyme activity of the in-house standard control. Then the absolute enzyme activities of the unknown samples were calculated by comparison with the in-house control sample, and expressed as U/mL.
Determination of glycaemic responses
The 54 subjects were ranked for their fasting and stimulated salivary amylase activities and averages of the resulting ranking coefficients were calculated. The highest and lowest ranked subjects were invited to further participate in the glycaemic response tests of this study. Some subjects were unable or unwilling to participate in this second phase. Consequently more medially ranked subjects were recruited in sequence of their ranking (high or low). Eventually, five in the low activity group and five in the high amylase activity group were available for comparing the glycaemic response to starch. The two experimental test foods comprised starch in 101.8 g of white bread (served with 200 mL water) and maltose in 62.5 g of rice malt syrup diluted with 200 mL water. These test foods each contained equivalent contents of hexose monomer, taking account of the relative equivalent monomer molecular weights of 168 for starch, 174 for maltose and 180 for glucose. A control glucose tolerance test using 55.6 g of glucose powder (Glucolin glucose supplement) dissolved in 200 mL of water was administered twice to each subject.
Glycaemic responses were tested on participants in the morning after an 8-h overnight fast. Participants received the test and control carbohydrates, in random order, with a minimum washout between each bolus of 4 days.
During the test with bread as the source of starch, each participant was requested to chew this and to swallow it over 15 min, together with 200 mL of water, which assisted ingestion. When subjects consumed one of the sugar solutions, they were advised to swish this thoroughly in their mouths, to mix it well with saliva, before drinking it at a constant rate over 15 min. Blood glucose levels were measured using the finger-prick method with a glucometer (Freestyle Optium glucometer, Abbott), immediately before consuming the test food, and at 15, 30, 45, 60, 90 and 120 min thereafter.
Sample size calculation
With the statistical level of significance set at 5 % and power at 80 %, 31 subjects were required, in order to evaluate the averaged salivary amylase with a desired precision of 4 U/mL, assuming a standard deviation of 11 U/mL . 50 subjects were recruited for sampling in order to account for potentially incomplete data.
Four subjects in the high amylase secretor group and four subjects in the low amylase secretor group were required to detect a difference of 112 mmol/L*120 min in AUC between the 2 groups with a standard deviation of 55 mmol/L*120 min as determined in Mandel’s study . To account for a possible drop-out, 5 subjects were recruited in each group.
Statistical analysis was carried out using SPSS Statistics version 18 (IBM, New York, US). Descriptive statistics were used to present the data mean and standard deviation. Comparisons between subjects in the low and high amylase groups were performed using a Mann Whitney test or Student T-test, as appropriate.