Reading Imperial Surrey Saturated Fat Cholesterol Intervention (RISSCI) Study. RISSCI-1 Blood Cholesterol Response Study
Phase: NA
Conditions: Lipids, Lipid Metabolism, Healthy
Sponsor: University of Surrey

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Reading Imperial* Surrey Saturated fat Cholesterol Intervention (RISSCI) Study 
RISSCI-1 Blood Cholesterol Response Study 
Study Protocol - Version 8, 16/7/18 
 
Brief background to the study 
Raised blood cholesterol (also referred to as blood LDL-cholesterol) is a major risk factor for 
developing heart disease. Dietary saturated fat is recognised as the main dietary component 
responsible for raising blood LDL-cholesterol, and reducing its intake has been the mainstay 
of dietary guidelines for the prevention of heart disease for over 30 years. However, there is 
very little evidence for a direct link between the intake of saturated fat and risk of dying from 
heart disease. One explanation for this, is that the link between saturated fat intake and 
heart disease is not a direct one, but relies heavily on the ability of saturated fat to raise 
blood LDL-cholesterol levels. This LDL cholesterol-raising effect of saturated fat is complex, 
and highly variable between individuals because of differences in the metabolism of dietary 
fat and cholesterol between people. While these differences in metabolism make it difficult to 
study how dietary saturated fat influences LDL-cholesterol in large numbers of people, they 
can be measured in the laboratory and used as biological markers to distinguish between 
people who respond well from those who will respond less well to moderate-fat diets which 
are lower in saturated fat. The main aim of this study is to measure the amount of variation in 
blood LDL-cholesterol in 150 healthy volunteers (75 at the University of Surrey and 75 at the 
University of Reading) in response to lowering the amount of saturated fat in their diet to the 
level recommended by the government for the prevention of heart disease. This 
collaborative project between the Universities of Reading and Surrey (‘RISSCI-1 Blood 
Cholesterol Response Study’), will allow us to investigate possible underlying causes for 
this variation in the blood cholesterol response in the whole group, and to identify two 
subgroups of men who show either a high or low LDL-cholesterol response to a reduction in 
dietary saturated intake. These men, will be provided with an opportunity to participate in a 
similar follow-up study (‘RISSCI 2’) that will require 36 men, 18 at the University of Surrey 
and18 at the University of Reading). In this follow-up study, the participants will be asked to 
repeat a similar study protocol as for RISSCI-1, and undergo more detailed measurements 
to determine how saturated fat is metabolised in the body (see section on ‘Retention of 
Participants’). The results of the RISSCI-1 study will be used to overcome the problems of 
setting dietary guidelines for whole populations, which are frequently inappropriate for some 
subgroups of people within that population. This will be achieved by the tailoring of dietary 
advice to those at higher risk of developing heart disease, and who stand to gain the 
greatest benefit to their health. *Note: Imperial College London is not involved in RISSCI-1. 
Study Objectives  
1. To recruit 75 middle-aged men from the local population in Surrey who meet our inclusion 
and exclusion criteria, using Surrey Clinical Research Centre’s (SCRC) data base of 
human volunteers.  
 
2. Invite eligible volunteers to a screening visit to ascertain their suitability to participate in a 
dietary intervention study (NB: >75 prospective volunteers will need to be screened until 
the target sample size of 75 is attained).   
  

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3. Obtain informed consent from eligible volunteers for their participation in the RISSCI-1 
Blood Cholesterol Response Study, and permission to contact them regarding 
participation in the follow-up study (see point 6 below).  
 
4. Undertake a dietary intervention study to examine the effects of two, 4 week diets that 
differ in their composition of fatty acids. The first diet (‘Diet 1’) will contain 18% of its total 
energy as saturated fatty acids (SFA), while the second diet (‘Diet 2’) will contain 10% of 
its total energy as SFA. Blood and stool samples taken at the beginning (week 0) and end 
of Diet 1 (week 4), and end of Diet 2 (week 8), will be analysed to measure blood LDL-
cholesterol, LDL-receptor gene expression (from isolated peripheral blood mononuclear 
cells) and other relevant blood and faecal* metabolites (* optional sample). The white 
blood cell buffy coat will also be isolated from the blood samples collected at the baseline 
visit to enable genotyping of relevant genes involved in the absorption and metabolism of 
dietary fat.  
 
5. To examine the data for evidence of associations between the changes in blood LDL- 
cholesterol, and the physical and biochemical characteristics of the participants as 
possible determinants of the variation in serum cholesterol response across the whole 
cohort (n=150). This will include measurement of a common genetic polymorphism in 
APOLIPOPROTEIN E, as an established determinant of variation in blood cholesterol in 
response to dietary SFA.  
 
6. To identify two subgroups of individuals whose blood LDL-cholesterol either responds  
(‘Responders’) or show little or no response (‘Non-responders’) on changing from Diet 1 
to Diet 2, for participation in the follow-up study (RISSCI-2), which will be conducted at 
the Universities of Surrey (n=18), Reading (n=18) and Imperial College London.  A 
separate ethics application will be submitted for RISSCI-2.  
 
  
Hypothesis                                                                                                                                         
In accordance with the variation in blood LDL-cholesterol response, that we and many others 
have reported previously following substitution of dietary saturated with unsaturated fats 
(RISCK2, DIVAS1), we hypothesise that consuming Diet 1 (a high saturated fat diet) for 4 
weeks followed by diet 2 (a low saturated fat/high unsaturated fat diet) for a further 4 weeks, 
will: 1) produce a spread of blood LDL-cholesterol responses that enables us to study 
associations between the participants’ baseline characteristics as possible determinants of 
the observed variation in blood LDL-cholesterol response, and 2) identify two distinct 
subgroups of individuals who either respond or show little or no response in their blood LDL- 
cholesterol. These distinct groups will be defined by the top and bottom 10% of change in 
the concentration of blood LDL-cholesterol in the subject group of 150.   
Criteria for the selection of participants 
Inclusion criteria: To remove the confounding effects of significant variance in blood 
cholesterol metabolism introduced by gender effects in a small cohort, the study will be 
restricted to middle-aged men (30-65 y) with a BMI of 19-32 kg/m2; fasting serum total 
cholesterol < 7.5 mmol/l and TAG < 2.3 mmol/l. No ethnicity restrictions will be imposed 

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however participants will be required to complete an ethnicity questionnaire, as it is known 
that this can influence lipid metabolism. 
Exclusion criteria: smokers, medical history of MI or stroke in the past 12 months; diabetes 
(defined as fasting glucose > 7.0 mmol/l) or other endocrine disorders; medication for 
hyperlipidaemia (e.g. statins) or prescribed antibiotics within the last three months; drinking 
in excess of 14 units of alcohol per week, anaemia (<130 g/L haemoglobin), or planning on a 
weight-reducing regime or taking any dietary supplements known to influence lipids/gut 
microbiota (e.g. plant stanols, fish oil, phytochemicals, natural laxatives, probiotics and 
prebiotics); unwilling to regularly consume study intervention products (butter/spreads, oils, 
dairy, snacks); or any other unusual medical history or diet and lifestyle habits or practices 
that would preclude volunteers from participating in a dietary intervention and metabolic 
study. 
Number of participants, recruitment methods and method for taking informed 
consent: The total sample size for the RISSCI-1 Blood Cholesterol Response Study is 150 
men; 75 men will be recruited at both the Universities of Surrey (UoS) and Reading (UoR), 
the latter of which will conduct an identical study to that performed at UoS. UoR will seek a 
favourable ethical opinion from its own University Research Ethics Committee. The sample 
size of 150 was chosen to provide the required number of responders and non-responders 
on changing from Diet 1 to Diet 2, to provide significant differences in our metabolic outcome 
variables between each of these subgroups in the follow-up study (RISSCI-2). On the basis 
of the highest and lowest 10% of change in blood LDL-cholesterol in a previous human 
intervention trial ‘DIVAS’1, the screening of 150 men will provide subgroups of 15 hyper-
responders and 15 hypo-responders with 20% drop-out rate, requiring 36 men in total), 
which corresponds to the predicted differences in outcome variables in our RISSCI-1 follow-
up study. From our previous trials, we predict that the distribution of blood LDL-cholesterol 
response will be similar at UoS and UoR, and thus provide equal numbers of high and low 
LDL-cholesterol responders at each site.          
Recruitment methods: All aspects of this study that involve the participation of human 
volunteers (screening, pre and post dietary intervention visits), will be undertaken within the 
SCRC at UoS. This is a world class research facility with MHRA Phase I Standard and 
Supplementary Accreditation to run clinical trials. It is fully staffed with study physicians, 
research nurses, and recruitment managers, and holds a large database of human 
volunteers who have registered as being willing to participate in clinical trials. Many of these 
volunteers have already been participants on previous studies, so are familiar with the 
SCRC, and taking part in intervention studies. After completion of the Medical & Lifestyle 
questionnaire and/or a brief telephone interview, volunteers who meet the inclusion and 
exclusion criteria for the study will be invited to attend the screening visit. Recruitment 
materials used for the purpose of recruitment from the SCRC database will be based on a 
standard template used by the unit. Separate recruitment material (a shorter email & 
alternative poster) will be used by members of the RISSCI research team for the purpose of 
advertising to staff and the wider community. Posters will be placed on designated poster 
boards, and HoD approval will be sought prior to email distribution.  
Method of taking informed consent: Potential volunteers invited to attend a screening visit 
will be sent the ‘Participant Information Sheet’ either by post or e-mail. This will explain the 
purpose of, and provide a brief description of the study, and explain why they have been 

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invited to volunteer. It will also explain what will happen if they choose to withdraw, and what 
the obligations and commitments of the participant are throughout the study. It will inform the 
participant of what happens to the data and samples, of any benefits and disadvantages of 
taking part in the study, what happens when the study stops and details about remuneration 
for their participation in the study. Finally, it will provide the contact details of persons they 
can contact if they have any questions, complaints or concerns about the study. If the 
volunteer accepts the invitation to attend the screening visit after reading the Participant 
Information Sheet, we will arrange the visit and go over the document with them and answer 
any queries. If they are happy to proceed, they will be invited to read and sign a ‘Consent 
Form’. The volunteer will sign the Consent Form in the presence of an independent witness, 
who will date and counter sign the Volunteer’s signature.  A copy of the Consent form will 
then be taken and given to the volunteer. 
Retention of participants for the follow-up study (RISSCI-2): The Consent Form contains 
a question that asks participants if they would be willing to be considered to take-part in a 
similar follow-up study. The Participant Information Sheet will explain that in the RISSCI-2 
study volunteers will be asked to repeat exactly the same two diets as in the RISSCI-1 study 
(Diet 1 followed by Diet 2) each for 4 weeks, and attend two short (~0.5-1 h) and two longer 
visits (~ 9 h) at the end of each diet, for more detailed measurements to determine how 
saturated fat is metabolised in the body. The longer visits will involve the volunteers being 
cannulated to obtain small, timed blood samples (~1ml) over 8 h, and the collection of urine 
and stool samples as in the RISSCI-1 study. It will be made clear to participants that they are 
under no obligation to take-part in this follow-up study, and that the stool and urine samples 
on this second study would not be optional.  
 
 Experimental design and methods to be used in RISSCI-1  
  

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Screening visit: Volunteers who meet the study inclusion and exclusion criteria (as 
determined by the Medical & Lifestyle questionnaire sent by e-mail or performed during the 
telephone interview), and have accepted the invitation to attend a screening visit, will be 
asked to fast for 12 hours and avoid drinking alcohol the evening before attending their visit 
to the SCRC. On the morning of their visit, (all appointments for visits to the SCRC will be 
made between 7.00-10.00am and be approximately 1 hour in duration), they will sign the 
Consent Form (as mentioned above), and have their body weight and height measured for 
their BMI, and following the measurement of their blood pressure, provide a sample of 
venous blood (14 ml) for the measurement of serum cholesterol, triacylglycerol (TAG) 
glucose, kidney and liver function tests (9 ml) and determination of the haemoglobin level 
and full blood count (5 ml). Suitable individuals will be invited to participate in the RISSCI-1 
Blood Cholesterol Response Study. Participants’ GPs will be notified regarding research 
participation and will be provided a copy of the screening blood test results;; it will therefore 
be up to the GP’s clinical judgement as to whether any action is required. The study has a 
sequential design in which diets will be consumed in the following order: Diet 1 for 4 weeks 
followed by Diet 2 for a further 4 weeks. This sequential intervention study design has been 
adopted by several studies, including our own Wellcome Trust funded ‘SATgene’ study 
(n=90)3, and FSA-sponsored ‘FLAVURS’ fruit and vegetable intervention (n=180)4. A double 
blind, placebo-controlled cross-over study cannot be adopted in this case, primarily because 
of the variation introduced by the manipulation of dietary fat, and returning subjects to their 
habitual diet during the wash-out period. Visits to the SCRC at UoS, will be made at the 
beginning (week 0) and end of Diet 1 (week 4) and Diet 2 (week 8).  
Intervention diets: To comply with current dietary recommendations, Diets 1 and 2 will 
contain 35% energy from total fat, with Diets 1 and 2 providing 18% and 10% total energy of 
SFA, respectively. The SFA-replacement fats will be mixture of poly (PUFA) and mono 
(MUFA)-unsaturated fatty acids. These diets will be consumed within the homes of free-
living participants, by the substitution of 40g of habitual fat, with either SFA-rich or 
MUFA/PUFA-rich cooking oils, spreads and snack foods, while maintaining their habitual diet 
(consistent intake of protein and carbohydrates, including dietary fibre). This will be achieved 
using a dietary exchange model developed for the ‘DIVAS’ study1, which replaced 9% total 
energy as SFA with MUFA/PUFA. This strategy has been successful in implementing large, 
complex dietary interventions at UoR and UoS (eg. DIVAS1, RISCK2, SATgene3, OPTILIP5). 
Replacement foods for the dietary exchange will be collected from the SCRC, and dietary 
compliance to the study protocol will be monitored every two weeks using tick sheets, either 
in person or over the telephone by the post-doctoral research dietitian (Dr Rona Antoni).  
Procedure: After the screening visit, eligible volunteers will be asked to complete a 4-day 
weighed dietary intake. In this pre-study period, participants will be contacted by telephone 
and given the opportunity to ask any further questions about the study and to arrange the 
date of the first intervention visit (referred to as the baseline visit or week 0). They will also 
need to collect from the SCRC, the study meal to consume the evening before the baseline 
visit, containers for the collection of the urine* and faecal* samples (*optional) and be given 
instructions on how to collect these samples. On the day prior to each study visit, they will be 
instructed to avoid alcohol, vigorous exercise and to consume the standard meal provided by 
the researchers before fasting overnight for 12 h (only drinking water during this time). 
Participants will be asked to bring along a fasted urine spot sample  (optional, collection the 
morning before study day, stored in cool bag and ice packs) and a stool sample (optional, 

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collected from the morning of the study day) to the study visit and will have their body 
weight, body composition (waist/hip circumferences via tape measure, percentage body fat 
and muscle mass using bioelectrical impedance), and blood pressure measured. 
Participants will also be asked to provide a brief 24-hour dietary recall, and to complete a 
physical activity questionnaire (Appendix XIV). Participants will also complete an ethnicity 
questionnaire on this initial baseline visit (Appendix XV). Participants will then provide a 
venous blood sample (50ml, equivalent to 3 tablespoons) for the measurement of blood total 
cholesterol, triacylglycerol, HDL-C (concentration/composition) and LDL-cholesterol, other 
markers of cardiovascular disease (e.g. inflammatory markers), for the isolation of white 
blood cell (WBC) buffy coat for the recovery of DNA for genotyping, and for the isolation 
peripheral blood mononuclear cells to measure LDL-receptors gene expression. Participants 
will then be supplied with their first set of foods for Diet 1, and instructed on how to 
incorporate these foods into their own diet. They will also be informed that they can collect 
foods from the SCRC when necessary and how to record their compliance to the study 
foods. After 4 weeks on this diet, participants will complete a 4-day weighed dietary intake 
before attending the SCRC for their second study visit, the procedure for which will be 
identical to the ‘baseline’ visit. Individuals will then be instructed on how to include the foods 
for Diet 2 in their daily diet for the next 4 weeks, and will be informed that they can collect the 
foods from the SCRC as before. They will also be provided with the standard meal and 
collection pots for the optional urine sample and stool sample to collect prior to the third 
study visit (week 8). At this final visit, the procedure will be identical to the study visit 
performed at weeks 0 and 4 of the study. Individuals identified within the top and bottom 
10% of change in fasting blood (LDL) cholesterol (week 8 minus week 4), will then be invited 
to participate in the RISSCI-2 follow-up study. Participants will be asked to complete an 
expenses form to enable them to be compensated for their travel, time and inconvenience 
(£120, plus an additional £20 for stool samples and £10 for urine samples; max total £150), 
following full completion of the RISSCI-1 Baseline Cholesterol Response Study. No payment 
will be provided to participants who withdraw from the study.  
It was decided to make the urine and faecal samples optional in order to minimise participant 
burden.  
Information on the collection retention, use and disposal of research data and 
measures in place to ensure confidentiality of personal data: Blood samples will be 
taken at screening, with blood and faecal samples collected at the baseline, week 4 and 
week 8 visits. Samples will be transported and analysed at the UoR.   
Ethical issues: There are no risks or reported side effects associated with the study foods 
in the intervention diets, as they are all normal components of the diet and commercially 
available in the supermarket. While we do not anticipate any adverse reactions to the study 
foods, food sensitivity and/or allergy will be recorded and avoided accordingly. All 
procedures will be performed by trained researchers, this includes venepuncture which will 
be performed according to a Standing Operating Procedure (SOP Ref. CPA-SOP-2). 
However, in case of an emergency (e.g. fainting, blood spillage) SCRC staff are trained in all 
relevant SOPs that describe in detail the actions that need to be taken by the researchers. 
Also, first aid is available, and physicians on-call in the SCRC when blood is being taken. In 
case of emergency, the research scientists will notify the SCRC staff who will provide first 
aid and, if necessary, contact University security to call the emergency services (999).  

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Consideration for working with other institutions (e.g. data protection): The RISSCI-1 
Blood Cholesterol Response Study will be undertaken at two sites, SCRC at UoS and Hugh 
Sinclair Unit of Human Nutrition at UoR, at which the study protocols are identical, with the 
exception of a difference in recruitment strategies and staffing. UoR will obtain a favourable 
ethical opinion from its own University Research Ethics Committee. Blood serum and stool 
samples collected at pre and post-dietary visits (baseline, weeks 4 and 8), PBMC and WBC 
buffy coats for isolation of DNA from the pre-diet (baseline) visit only, will be anonymised 
with a study code to ensure confidentiality of personal data. The names and codes of 
participants at each research site will be stored securely on the University server at both the 
UoR and UoS, access to which will be password protected and only accessible at each site 
by the respective Principle Investigators (Professors Griffin & Lovegrove) and post-doctoral 
research assistants (Drs Antoni & Koutsos). After collection of the screening samples at 
UoS, samples will be stored briefly at room temperature or 4oC (<30 min) prior to 
centrifugation (1700 g x 15 min) and aliquotting of samples into appropriately labelled tubes. 
Samples will be stored at -20°C until transportation at this temperature by courier to UoR for 
analysis. All of the study intervention visit samples will be processed at each of the study 
sites according to the agreed study protocols, and stored at the appropriate temperature until 
sample transfer and analysis on completion of the study. A Material Transfer Agreement 
(MTA) will be in place for the transfer of samples between UoS and UoR, in accordance with 
the Human Tissue Act 2004. All data produced from these samples will be stored on a 
password-protected central data base held on the secure University servers at UoS/UoR, 
and handled in accordance with the Data Protections Act 1998. After analysis, all samples 
will be stored frozen (-20oC or -80 oC) for a maximum period of 36 months, and then 
destroyed.   
Study evaluation and statistical analysis 
Dietary compliance to the study protocol will be evaluated on a fortnightly basis in person or 
over the telephone by using product tick-sheets and monitoring of body weight. Participants 
will be asked to self-report their weight, measured on home scales on day one and half way 
through of each four-week dietary intervention period. Any changes in body weight of at least 
1 kg will be reviewed by the study researchers and further dietary advice will be given to the 
participants as required. Data from the metabolic study will be checked for normality and 
analysed using a mixed factor ANOVA with repeated measures to determine the impact of 
the dietary interventions on our outcome measures. The study will be registered as a clinical 
trial on ‘Clinical Trials.gov’ prior to the recruitment of the participants for the RISSCI-1 Blood 
Cholesterol Response Study.  
 
References 
1. Vafeiadou K et al (2015) AJCN 102, 40-8.   
2. Jebb SA et al (2010) AJCN 92, 748-58.  
3. Lockyer S et al (2012) Br J Nutr 108, 1705-13. 
4. Macready AL et al (2014) AJCN 99, 479-89. 
5. Griffin MD et al (2006) AJCN 84, 1290-8. 
 

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