Impact of Inulin on Production of Phenolic Acids From Tomato Onion and Lovage Soup
Phase: NA
Conditions: Healthy Adults
Sponsor: University of Glasgow

 
 
 
School of Medicine, Dentistry and Nursing 
 
 
Study Protocol and Statistical Analysis Plan 
 
Impact of Inulin on Production of Phenolic Acids from Tomato Onion and Lovage Soup 
 
 
 
 
 
07/02/2018 
 
 

 
 
 
 
 
Study Protocol and Statistical Analysis Plan 
Study design: Acute human bioavailability studies, with a cross-over design.  In some cases, using 
stable isotope labelled enriched polyphenols added as a spike of enriched parent compound.  
Arm/ group information 
Non blinded - crossover 
1. 
Soup containing tomatoes, red onion and lovage (polyphenol rich)   (TOL) 
2. 
Inulin (Non digestible carbohydrate; NDC)   (INU 
3. 
Mixture of soup and inulin  (TOL+INU) 
Each soup will also contain 1g paracetamol to allow estimation of effects on gastric emptying; breath 
hydrogen will be collected to determine any effects on mouth to caecum transit time. 
 
Wash out duration between feeding studies: one week 
 
Recruitment:  Self-reported healthy adults, 20-70y  any gender 
Exclusion criteria: antibiotic use within the last 3 months, identified gastro-intestinal diseases, on 
prescribed medication other than the contraceptive pill, individuals who are pregnant or 
breastfeeding. Individuals who have been diagnosed as anaemic, as well as those who are allergic to 
any food, or paracetamol. 
 
Intervention recipes: Polyphenol rich soup containing cherry tomatoes (300g) red onion 100g and 
lovage 20g. (includes quercetin and rutin), using enriched deuterium labelled onions and lovage 
grown at the James Hutton Institute in Dundee, UK in some cases.  
The NDC (fibre): inulin, was chosen based on previous in-vitro work, at a dosage of 10g per drink.  
 
Background diet:  Volunteers will maintain a low polyphenol/ low fibre diet for two days before each 
study day and until completion of the 24 hour urine collection started on the study day. 
 
 
 

Procedures on each study day 
Procedure 
Occasions 
Estimated 
Time taken 
Details 
Cannulation 
and blood 
collection 
3 
12-hours 
Participants will be fitted with a cannula to facilitate 
blood sampling. Blood samples will be taken at 
baseline (6ml). After the start of the intervention, 
blood will be collected every 15 minutes (6mL) for 
the first two hours and every 1 hour thereafter up to 
8 hours. The cannula will be removed eight hours 
after the start of the test and the subject will be 
allowed to leave the unit. The subject will return 
with a 24-hour urine sample, and at 24 hours a 
blood sample (6mL) will be collected by 
venipuncture.  Participants may provide a faecal 
sample at 24 hours. 
 
Feeding 
study 
3 
5 min 
Participants will be given the test soup (polyphenol 
rich soup alone, inulin alone, or mixed polyphenol 
and inulin soup) to eat. 
Urine 
collection 
after the 
study 
3 
24 hours 
Participants will be provided with containers for urine 
collection, which will be collected during the test day 
and over the next 18 hours split into 0-2 hours, 2-5 
hours, 5-10 hours and 10-24 hour collections. Water 
will be provided ad libitum throughout the day, with 
encouragements to drink every 30 minutes. 
Breath 
hydrogen 
monitoring 
3 
>10 
Breath hydrogen will be monitored via expelled 
breath at regular 30 minutes intervals until a rise in 
hydrogen has been observed. 
Lunch at the 
unit 
3 
30 minutes 
Participants will be given a low fibre low polyphenol 
meal. The amount eaten will be recorded.  
Palatability 
testing 
3 
30 minutes 
Participants will be asked to fill a short questionnaire 
regarding the palatability of the soup within 30 
minutes of ingestion. 
 
 
 

Statistical analysis Plan 
This study will test the hypothesis that combination of polyphenolics and inulin in a soup made with 
Tomatoes, onion and lovage will increase the phenolic acid urinary output in comparison to the 
same soup without inulin.  
 
Sample size:  
Our previous work with orange juice in healthy sedentary females led to an excretion of 598±144 
umoles/24h of phenolic acids in urine.  To detect a difference in phenolic acid output by 20%. using a 
matched pair, one-side test, with power at 80% and alpha at 5%, we require 15 participants in this 
bioavailability study. 
 
Randomization of participants: 
The participants will be randomized to the order of diets using opaque envelopes with test food 
order enclosed.   
Test for normality: 
Data will be tested for normality by the Shapiro Wilk Test, if non parametric data will be log 
transformed before statistical analysis.  
Primary outcome:    
The primary outcome is total urinary phenolic acid content over 24 hours before and after the test 
meal (umoles/L, mg/L, umoles/day).   The effect of inulin will be tested by repeated measures 
analysis of variance (ANOVA) followed by post hoc Bonferroni to correct for multiple testing. 
Secondary outcomes:  
Individual and total phenolic acid content in urine at 0-2hours, 2-5, hours. 5-10 hours and 10-
24hours. (umoles/L, mg/L, umoles/day 
These will be compared by repeated measured ANOVA and post hoc Bonferroni  to correct for 
multiple testing . 
Plasma glucose levels (mmoles/l) 
This will be measured at baseline before the meal and then every 15 mins for 2 hours then every 1 
hour for 6 hours .   The data will be compared by repeated two way repeated measures ANOVA as 
changes over the day and also converted to area under the curve and compared between arms by 
repeated measures ANOVA. 
Plasma insulin (mIU/ml) 
This will be measured in plasma every 15 mins for 2 hours then every 1 hour for 6 hours .  The data 
will be compared by repeated two way repeated measures ANOVA as changes over the day and also 
converted to area under the curve and compared between arms by repeated measures ANOVA. 
 

Satiety hormones GLP-1 and PYY (picomoles/l) 
These will be measured in plasma every 15 mins for 2 hours then every 1 hour for 6 hours .  The data 
will be compared by repeated two way repeated measures ANOVA as changes over the day 
Gastric emptying (T1/2 mins) 
Paracetamol levels in plasma will be measured over 240 mins to estimate gastric emptying rate.  The 
data will be analysed as area under the curve.  The t1/2 for gastric emptying between arms will be 
compared by repeated measures ANOVA followed by Bonferroni to correct for multiple testing  
Mouth to caecum transit time (hours/ mins) 
Breath hydrogen samples will be collected at baseline and every 30 mins.  Mouth to caecum transit 
time in minutes will be estimated as the first sustained rise in breath hydrogen.  Difference between 
arms will be repeated measures ANOVA followed by Bonferroni to correct for multiple testing  
Faecal phenolic acids (umoles/g, mg/g)  
If sufficient faecal sample has been provided, phenolic acids will be measured.  The amounts will be 
compared by repeated measures ANOVA and Bonferroni correction.