The Effect of Millet-based Muffins on Glycaemic, Insulinemic Response and Gastric Emptying in Pre-diabetic Adults
Phase: NA
Conditions: Healthy, Pre Diabetes
Sponsor: Oxford Brookes University

CONFIDENTIAL 
 
March 2017 
 
 
1 
Protocol for glycaemic response, insulin response, gastric emptying and satiety testing (3 hours) 
 
 
Aim 
The study aims to assess the glycaemic response (GR), insulinaemic response (IR) and gastric 
emptying (GE) time after the consumption of a millet-based muffin in pre-diabetic and healthy 
participants in order to establish whether this lowers GR and IR compared to the consumption of a non-
millet-based, control muffin. 
 
PROPOSED METHOD:   
 
This study will investigate the effect of polyphenol-rich millet-based muffin on GR, IR and GE in 
healthy and pre-diabetic participants (people with prediabetes have blood glucose levels that are 
higher than normal but not yet high enough to be diagnosed as diabetes - the normal fasting blood 
glucose level is below 6.1 mmol/l or 108 mg/dl).  
 
After potential participants have read the PIS and signed the consent form, they will be screened 
during the first visit to measure fasting blood glucose (FBG; fasting blood glucose should be between 
6.1 to 6.9 mmol/l for pre-diabetic participants and < 6.1 mmol/l for healthy participants ).If eligible for 
the study, body weight and height and blood pressure will be taken in the Functional Food Centre. If 
the fasting blood glucose test is less than 6.1mmol/l (participants will be informed of their fasting blood 
glucose result and given  the choice either to do Oral glucose tolerance test (OGTT) to check for 
Impaired Glucose Tolerance (IGT), particpate in the healthy group  or to be excluded). If the 
participant agrees to continue and has one or more of the pre-diabetes inclusion criteria,they can 
complete an oral glucose tolerance test (OGTT) over 2 hours.( An oral glucose tolerance test involves 
taking a fasting sample of blood and then taking a very sweet drink containing 75g of glucose. After 
having this drink participants need to stay at rest until a further blood sample is taken after 2 hours.) If 
their 2 h blood glucose result is between 7.9 to 11.0 mmol/l, they will be considered eligible to 
participate in the study as a prediabetic participant, and will be asked to come for a subsequent visit in 
order to begin the study (and therefore the first visit will act as a ‘screening visit’) (please, see chart 
below for more detail).If the participant does not have IGT, they have the option to take part in the 
study as a healthy participant.  
 
 

CONFIDENTIAL 
 
March 2017 
 
 
2 
Interested participants (pre-diabetic group) will be invited for screening test to check their 
fasting blood glucose level and will go through the following protocol. Testing is completely 
voluntary to the participant:  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening visit 
Fasting blood glucose test (FBG) for all volunteers 
If the result is less than 6.1 mmol/l and 
participant has one or more from criteria in the 
pre-diabetic inclusion criteria. 
If the result of FBG is between 6.1 
to 6.9 mmol/l                       
Participants will be informed of their fasting blood glucose result and have the 
choice either to be excluded or to do an  oral glucose tolerance test (OGTT). 
Participants will be informed of their fasting 
blood glucose result and  their eligibility to 
participate in the study  will be confirmed. 
Moreover,  It will be made very clear to the 
participant that the diagnosis of pre-diabetes 
is never made on the basis of a single 
abnormal blood glucose value. 
To avoid any anxiety, they will be given the 
choice either to participate in the study 
directly or to seek GP advice if they wish and 
then will be allowed to join the study. 
 
Why OGTT? If participant have one or more of the inclusion criteria and FBG test is 
less than 6.1 mmol/l, they will be asked to do OGTT in the near future to confirm 
whether they are prediabetic or not (Diabetes,UK).OGTT , can show if your body is 
having problems processing glucose. 
If 
the 
participant 
disagree 
to 
continue they 
will 
be 
excluded and 
a snack will be 
given as thank 
you. 
If the participant agree to continue, Oral glucose 
tolerance test (OGTT) at 2 hours will be carried out.  
If the result at 2 
hours 
is 
less 
than 7.8 
mmol/l 
the 
participants 
will be excluded 
from the study and 
snack will be given 
as thank you  
OR  the participant 
can take part in the 
healthy group if 
eligible and meets 
the other inclusion 
criteria. 
. 
 
If the result at 2 hours is between 7.9 to 
11.0 mmol/l, participants will be eligible 
to take part in pre-diabetic group and 
and they will be informed of their OGTT 
result 
To avoid any anxiety, they will be given 
the choice either to participate in the 
study directly or to seek GP advice if they 
wish and then will be allowed to join the 
study. 
 
If the participant agree to participate       
The following measurment will be taken: 
 
1. 
 Anthropometric measurement 
(weight, height and BMI), Blood 
pressure measurement (in first visit 
only). 
2. 
 GR, IR & GE tests (in both visits). 
 
Then, the  2nd visit will be booked  
 
Note: 2 sessions in total 
 
If the participant agree to participate      
Then, 2 visits will be booked and snack 
will be given as thank you. 
The following measurment will be taken: 
 
1.  Anthropometric measurement (weight, height and 
BMI), Blood pressure measurement (in first visit only). 
2.  GR, IR & GE tests (in both visits). 
 
Note: 3 sessions in total 
 

CONFIDENTIAL 
 
March 2017 
 
 
3 
1- Glycaemic response (GR) 
The method to be used is adapted from that described by Brouns et al. (2005) and is in line with 
procedures recommended by the FAO/WHO (1998) and ISO 26642:2010 guidelines (ISO 26642:2010 
Food products – Determination of glycaemic index). 
 
The control and millet muffins will be administered to 15 pre-diabetic participants and 15 healthy 
participants in a two-arm randomised, repeated measure, crossover design. The sample size is based 
on previous research conducted within the Functional Food Centre and has sufficient power to detect 
differences in blood glucose and insulin levels. All participants will consume control muffin (wheat) or 
test muffin (millet) in random order on separate occasions with at least one-day between each test 
and any day during the recruitment periods.On the evening prior to a test, the participant will be asked 
to avoid the consumption of caffeine, alcohol and nicotine, to avoid unusual, strenous exercise and to 
fast for 12 hours (overnight).  At the beginning of the first test session, the following measurements 
will be taken: 
• Body composition (using the Tanita BC-418MA segmental body composition analyser).  
• Blood pressure (using an automated sphygmomanometer) - if blood pressure is higher 
than normal, they will be advised to contact their GP for further tests if necessary (high blood 
pressure is considered to be 140/90mmHg or higher)                                 
• It will take approximately 15 minutes to complete these measurements. 
 
Blood samples will be taken at -5 and 0 min before consumption of the control/test muffin and at 15, 
30, 45, 60, 90,120,150 and 180 min after starting to eat. Finger-prick blood samples will be taken 
using a single-use lancing system (Unistick 3, Owen Mumford, Woodstock, UK).  Blood glucose will be 
immediately measured using an automatic blood glucose analyzer (Glucose 201+, Hemocue AB, 
Sweden).  The accuracy of the analyzer will be checked daily using control solutions.  Each 
measurement requires only 5 µl blood, therefore the finger-prick will be small, with minimal discomfort, 
and the participants will not experience any negative consequences except for a possible slight 
bruising on finger tips. All blood samples will be destroyed immediately after reading and recording, 
according to Standard Operating Procedures. For each product, the incremental AUC, ignoring area 
beneath the baseline, will be calculated geometrically (FAO/WHO, 1998) and will include the area 
above the fasting level only; any area beneath the fasting level will be ignored. 
 
Satiety 
Immediately before each blood sample, participants' subjective feelings of satiety/hunger will be 
recorded on 7-anchored bidirectional scales. 
 
2- Insulinaemic response  
The determination of insulin will be undertaken at the same time as the blood glucose measurements 
(procedure is as given below). 

CONFIDENTIAL 
 
March 2017 
 
 
4 
1. Finger-prick blood samples will be taken using a single-use lancing system (Unistick 3, Owen 
Mumford, Woodstock, UK). For  insulin, blood samples will be taken at the same time points 
and using the same finger-prick for  glycaemic  response. Each insulin measurement requires 
300 µl blood.  The finger-prick will be small, with minimal discomfort, and the participants and 
the participants  may have some bruising in thier fingers. Samples will be collected in EDTA-
coated tubes and kept on ice and will be centrifuged and the plasma frozen at -40°C until 
analysis. 
2. Plasma insulin will measured by immunoassay (Cobas e411, Roche, UK). 
3. The IR will be calculated geometrically by measuring the incremental AUC, ignoring the area 
beneath the baseline (FAO/WHO, 1998) and only include the area above the fasting level. 
 
3- Gastric emptying 
100 mg of 13C sodium acetate will be added to the muffins (control and millet) in order to measure 
gastric emptying rate. 13C is a naturally abundant stable isotope of carbon that is completely safe. 
Sodium acetate is regularly used in the food industry as seasoning. Breath samples for measurement 
of gastric emptying will be taken by blowing into a small glass tube through a straw. They will be 
collected every 15 minutes for 4 hours after breakfast (in the same time points of  glycaemic  and 
insulinaemic response plus at, 210 and 240 minutes).  
 
Statistical Analysis 
Data will be analysed using Microsoft Excel 2010 and SPSS 21.0 . Means, standard deviations, 
descriptive statistics and repeated measures Anova will be used to determine differences in glycaemic 
and insulinaemic response and gastric emptying between the control muffin and  millet muffin.  
 
References: 
 
Brouns F, Bjorck I, Frayn KN, Gibbs AL, Lang V, Slama G and Wolever TMS. Glycaemic index 
methodology. Nutr Res Rev 2005;18:145-171. 
 
FAO/WHO. Carbohydrates in Human Nutrition. Rome: FAO, 1998 
 
 
ISO 26642:2010 Food products – Determination of glycaemic index.