Protocol for a randomized controlled trial comparing saturated and unsaturated fat sources of a ketogenic diet (KETO-IM) investigating cardiovascular, inflammatory and immune function outcomes in adults living with overweight and prediabetes or type 2 diabetes in Edmonton, Canada

Abstract

Background

Among the general public, the ketogenic (KETO) diet is popular for weight loss and may also support glycemic control in people with type 2 diabetes (T2D). Obesity and T2D have both been associated with diminished immune responses, increasing the risk of infection. KETO diets are often high in saturated fats (SFA), which may increase cardiovascular disease (CVD) risk as well as inflammation. Substituting SFA for unsaturated fats (UFA) may improve these outcomes. This study aims to compare lipid measures, glycemia, inflammation, and immune function in people with, or at risk of developing, T2D after following KETO diets emphasizing SFA versus UFA versus a low-fat standard of care diet (LFD) for 6 months. We hypothesize that (1) circulating triglycerides (TG) will be reduced more in the two KETO groups relative to the control LFD and (2) low-density lipoprotein (LDL-C) will be lowered to a greater extent with higher UFA consumption relative to KETO rich in SFA.

Methods

The KETO-IM study is an open-label, single-center, three parallel-arm, superiority, randomized controlled trial (RCT). It will enroll adults aged 18 to 70 years with body mass index (BMI) > 23 kg/m² (Asian and South Asian), or > 25 kg/m² (non-Asian) and glycated hemoglobin (HbA1c) > 5.7%, recruited by the study team from the community in Edmonton, Canada and surrounding area. Participants (175 total) will be allocated to three groups in a 1:1:1 ratio with evaluation endpoints at three and six months compared to baseline. The primary outcomes assessing CVD risk, will be plasma LDL-C and TG. Secondary outcomes of the trial include glucose metabolism, inflammation and immune function. Among the secondary outcomes, comparison of immune function will be a novel focus.

Discussion

This trial will directly compare the effects of fat sources in a KETO diet to a control LFD on cardiometabolic risk, inflammation and immune function in adults of both sexes. The results of this trial can be used to guide clinical advice to patients seeking to improve lipid and glycemic control while, for the first time, comparing the effects of KETO diets on immune response.

Trial registration

ClinicalTrials.gov, NCT05681468. Registered on 12 December 2022, https://clinicaltrials.gov/study/NCT05681468?titles=keto-im&rank=1. First enrollment September 18, 2023.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13063-026-09501-0.

Administrative information

Title {1}	Protocol for a randomized controlled trial comparing saturated and unsaturated fat sources of a ketogenic diet (KETO-IM) investigating cardiovascular, inflammatory and immune function outcomes in adults living with overweight and prediabetes or type 2 diabetes in Edmonton, Canada
Trial registration {2a and 2b}	ClinicalTrials.gov, NCT05681468. Registered 12 December 2022, https://clinicaltrials.gov/study/NCT05681468?titles=keto-im&rank=1
Protocol version {3}	Pro00123687, Version 4
Funding {4}	Funding for this study has been provided by Alberta Canola and Results Driven Agriculture Research (grant #2022F156R). Research equipment and lab facilities are paid for by the grant funding received. This grant funding also includes an annual stipend amount for a graduate student on the project, as well as food and supplement costs for ingredients supplied to participants Student stipend and fellowship funding has been received from the Canadian Institutes for Health Research, Natural Sciences and Engineering Research Council of Canada, Social Sciences and Humanities Research Council, Alberta Diabetes Institute, Lorna Bradley Diabetes Research Endowment, and the University of Alberta
Author details {5a}	Jodie Harbarenko, BSc (Honours), RD CDE, Department of Agricultural, Food and Nutritional Science. Mailing address: Human Nutrition Research Unit, Li Ka Shing Centre for Health Research Innovation. University of Alberta, Edmonton, Alberta, T6G 2E1. Email: jharbare@ualberta.ca Dr. Trina Gartke, MD, PharmD. Department of Medicine, 13–103 Clinical Sciences Building, University of Alberta, Edmonton, Alberta, T6G 2B7. Email: tgartke@ualberta.ca Paulina Blanco Cervantes, MSc. Department of Agricultural, Food and Nutritional Science. Mailing Address: 2-021H Human Nutrition Research Unit, Li Ka Shing Centre for Health Research Innovation. University of Alberta, Edmonton, Alberta, T6G 2E1. Telephone: 1–780-492–9506. Email: blancoce@ualberta.ca Yasaman Mojibi, BSc., Department of Agricultural, Food and Nutritional Science. Mailing address: Human Nutrition Research Unit, Li Ka Shing Centre for Health Research Innovation. University of Alberta, Edmonton, Alberta, T6G 2E1. Email: ymojibie@ualberta.ca Alexander Makarowski, Department of Agricultural, Food and Nutritional Science. Mailing address: Human Nutrition Research Unit, Li Ka Shing Centre for Health Research Innovation. University of Alberta, Edmonton, Alberta, T6G 2E1. Email: amakarow@ualberta.ca Dr. Caroline Richard, PhD RD. Department of Agricultural, Food and Nutritional Science. Mailing address: 4−002G Li Ka Shing Centre for Health Research Innovation. University of Alberta, Edmonton, Alberta, Canada, T6G 2E1. Telephone: 1–780-248–1827. Email: cr5@ualberta.ca Dr. Catherine Chan, PhD. Department of Agricultural, Food and Nutritional Science. Mailing address: 4-126A Li Ka Shing Centre for Health Research Innovation. University of Alberta, Edmonton, Alberta, Canada, T6G 2E1. Telephone: 1–780-492–9939. Email: cbchan@ualberta.ca
Name and contact information for the trial sponsor {5b}	Brittany Visscher, Research Director, Alberta Canola. Telephone: 587–525–5937; email: brittany@albertacanola.com Trevor Prout, Research Program Manager, Results Driven Alberta Agriculture. Email: Trevor.Prout@rdar.ca
Role of sponsor {5c}	The funders have no involvement in the study design or the collection, analysis, and interpretation of data. Decisions regarding report writing and publication of data are made solely and independently by the study team. Salaries for the primary investigators and research coordinator are provided by the University of Alberta

Introduction

Background and rationale {6a}

Diabetes occurs when the body becomes unable to regulate blood glucose properly, either due to insulin resistance or an inability to produce enough insulin (usually both), leading to hyperglycemia [1]. Type 2 diabetes (T2D) now affects more than 56 million people in North America and over 500 million people worldwide [2]. This growing health problem necessitates that health professionals and researchers find solutions for appropriate treatments, including medical nutrition therapy.

The very low carbohydrate diet, also called a ketogenic (KETO) diet, is based on a macronutrient content of < 10% of total energy intake from carbohydrates, or < 50 g daily. The KETO diet has been used therapeutically for many years for pediatric epilepsy [3], and is gaining interest as a treatment for T2D as it improves blood glucose regulation following the reduction in carbohydrate intake [4]. However, KETO diets are often high in saturated fats (SFA), which may increase low-density lipoprotein cholesterol (LDL-C) and systemic inflammation, which are cardiovascular disease (CVD) risk factors [5]. Previous research documents conflicting results of high SFA intake for certain cardiovascular outcomes, including coronary heart disease and stroke. In 2015, a Cochrane review, including 15 randomized-controlled trials (RCTs), [6] reported that reduced SFA intake was associated with a reduction in risk for cardiovascular events (risk ratio (RR) 0.83; 95% confidence interval (CI) 0.70 to 0.98). A narrative review by Fattore and Massa, examining research conducted internationally, concluded that although SFA intake was associated with higher incidence of coronary heart disease and cardiovascular events, there was no association with all-cause mortality [7]. DeSouza et al. [8] found no association between SFA intake and cardiovascular outcomes (relative risk 0.97, 95% CI 0.84 to 1.12) in their review of 34 studies with international data, but stated the methods of the studies reviewed, and their findings, were heterogeneous. Fattore and Massa [7] also report that although some long-term cohort studies find links between higher intakes of SFA and cardiovascular outcomes, there is limited evidence from RCTs to support these findings. However, the types of foods consumed as fat sources might confound results. Thus, whether a KETO diet should be recommended for people with CVD risk remains debatable.

The existing experimental data support that consumption of foods rich in specific fatty acid classes modulate upstream risk factors for CVD. For example, in young adults, replacing SFA with monounsaturated fats (MUFA) for 2.5 weeks significantly decreased total cholesterol and LDL-C. High-density lipoprotein cholesterol (HDL-C) also decreased but to a lesser extent (12% vs 15% vs 4%, respectively) [9]. Compared with diets higher in SFA and cholesterol, the United States National Cholesterol Education Program Step I (≤ 30% total energy as fat, ≤ 10% of energy as SFA) and Step II (≤ 7% of energy as SFA, dietary cholesterol ≤ 200 mg/day) diets improved lipids and other CVD risk factors [10, 11]. Moreover, long-term prospective trials found significant reduction of CVD risk when SFA were replaced by polyunsaturated fats (PUFA) or MUFA [12]. Inflammation is an independent risk factor for CVD that can be induced by feeding SFA via a variety of intracellular mechanisms [13] whereas unsaturated fatty acids (UFA), especially omega-3 PUFA, attenuate such effects [14, 15]. Our previous data from the NutrIMM study also indicate that individuals living with obesity and T2D have impaired immune function (e.g., reduced T cell response to challenge) [16]. Both inflammation and insulin resistance in obesity have been proposed as major factors that negatively affect immune function [17]. Therefore, substituting SFA with UFA in the context of a KETO diet might counteract some of the obesity-related immune dysfunction [18].

Canola oil is high in MUFA (oleic acid) and PUFA (linoleic, linolenic acid) and has received USA Food and Drug Administration approval for specific health claims related to cholesterol-lowering effect when substituted for SFA [19]. For people with T2D, replacing dietary SFAs with sources of MUFA and PUFA also mitigates several CVD risk factors, including lowering triglycerides (TG) and increasing HDL-C [20]. Alpha-linolenic acid, an omega-3 fatty acid, is also associated with reductions in C-reactive protein (CRP), a marker of systemic inflammation [21, 22].

There have been few rigorous comparisons of the sources of fat in KETO diet studies. One trial [23] compared a low-carbohydrate diet (LCD) to a Mediterranean (MED) diet high in MUFA and low-fat diet (LFD), but did not match for fat intake. Nutrient analysis found that the MED diet group consumed 33% of total energy as fat at up to two years follow-up vs 39% for the LCD group and 30% for LFD. Although the LCD participants initially followed a KETO diet-like carbohydrate restriction, they were transitioned to a low carbohydrate classification over time, with up to 120 g of carbohydrates daily. Ultimately, only 8.3% of the LCD group exhibited ketosis at the two-year follow up. At one and two years, MUFA intake in the MED diet group increased significantly from baseline whereas in the LFD or LCD groups, MUFA intake remained stable. SFA intake was significantly higher in the LCD (12.3% of energy intake) than either MED diet or LFD groups (both 9.6% of energy intake) at 2 years. The MED diet had a superior glycemia lowering effect only in participants with diabetes [23], thus it is hypothesized that MUFA may have benefits for improving insulin sensitivity when replacing SFA but whether this is true in non-diabetic people is unclear. Because canola oil has similar MUFA content to olive oil (as used in the MED diet), we predict that it should also be superior to diets higher in SFA for glucose management.

Objectives {7}

This study aims to compare primary outcomes of LDL-C and TG in people with, or at risk of developing, T2D after following KETO diets emphasizing SFA (KETO-Sat), UFA through the use of canola oil (KETO-Can) or LFD for six months. Secondary outcomes include indicators of glycemic regulation, inflammatory markers, and the immune response. Analysis of the primary and secondary outcomes will be conducted at baseline, three months and six months. We will compare factors influencing adherence to diets and compliance by assessing diet records, presence of ketosis, and fatty acid composition of plasma and membranes of circulatory cells as exploratory outcomes. The primary hypotheses are that (1) circulating TG will be reduced more in the two KETO groups relative to the control LFD and (2) LDL-C will be lowered to a greater extent in the Keto-Can group relative to the Keto-Sat group.

Trial design {12}

KETO-IM is an open-label, three-arm, parallel group, superiority, RCT conducted at a single site with 1:1:1 allocation ratio.

Methods: Participants, interventions and outcomes

Study setting {9}

This study will be conducted at the Human Nutrition Research Unit (HNRU) of the Alberta Diabetes Institute (ADI) at the University of Alberta, Edmonton, Alberta, Canada. Research study visits and blood collections will be conducted at the HNRU. Participants will be recruited from the Edmonton region. The target population is adults living with overweight/obesity and at risk of or diagnosed with T2D.

Eligibility criteria {10}

Inclusion criteria: (1) Adults aged 18–70 y; (2) Body mass index (BMI) > 23 kg/m² (Asian and South Asian), or > 25 kg/m² (non-Asian) and glycated hemoglobin (HbA1c) ≥ 5.7% at screening/baseline; (3) Must be weight stable within the last three to six months; (4) Individuals with a prior history of CVD, renal disorders, endocrine disorders will not be excluded, but will be further assessed by the research team physician for safety of participation in the study; (5) Women with an irregular menstrual cycle will be further assessed to determine cause and eligibility.

Exclusion criteria: (1) Individuals with specific nutritional habits preventing them from following an allocated diet; (2) Documented history of an eating disorder; (3) Pregnant women or those who plan to become pregnant during the study duration; (4) Individuals with type 1 diabetes; (5) People on dialysis or those recommended to follow a low protein diet based on glomerular filtration rate; (6) Genetic disorders: familial history of hypertriglyceridemia or hypercholesterolemia; (7) Individuals undergoing gender reassignment procedure (until hormone status is stabilized); (8) Individuals with history of diabetic ketoacidosis; (9) For safety purposes, other individuals may be excluded if unstable health conditions are present; (10) Specific allergies to diet ingredients; (11) Unwilling or unable to comply with any study requirements or provide informed consent.

Study team criteria: (1) A clinical endocrinologist (MD) will screen participants to confirm eligibility; (2) Licensed phlebotomists will conduct blood draws at study visits; (4) Registered Dietitians (RD) with current Certified Diabetes Educator designation will provide nutritional counselling for all participants living with T2D; (4) All study team personnel will be trained and follow standard operating procedures for use of all equipment during study visits, including blood pressure machines and point of care devices.

Who will take informed consent? {26a}

After providing oral and written information about the trial according to an approved ethics protocol (Pro00123687), including its voluntary nature, the study coordinator or graduate students will obtain written consent from eligible participants. All personnel involved in obtaining consent will have ethics training.

Additional consent provisions for collection and use of participant data and biological specimens {26b}

The trial has provision for collection of fecal samples for an ancillary study of the effects of the intervention diet formulations on the gut microbiome. This aspect of the trial is optional for participants. Those willing to participate sign explicit informed consent for collection of stool samples.

Interventions

Explanation for the choice of comparators {6b}

The three comparator groups include:

1. KETO-Can: KETO diet supplemented with canola oil (high in MUFA and omega-3 fatty acids) with 60–70% of total energy intake from fat.

2. KETO-Sat: KETO diet supplemented with butter and coconut oil (high in SFA) with 60–70% of total energy intake from fat.

3. Low Fat: supplemented with whole grain foods including pasta or brown rice, and oatmeal (high in fibre) with up to 30% of total energy intake from fat.

The KETO diet is popular for weight loss and is gaining interest as a treatment for T2D [4] but are often high in SFA, which may increase LDL-C and exacerbate other CVD risk factors, such as inflammatory mediators [5]. Substituting a UFA source, such as canola oil, for SFA may improve these outcomes. The third comparator, LFD is considered the standard of care by many diabetes organizations [24, 25].

Intervention description {11a}

The LFD group will aim to achieve 30% of total energy intake from fats (< 10% as SFA), 50% of energy from carbohydrates (with emphasis on whole grains, including oatmeal, whole grain pasta and brown rice) and 20% from protein (mainly lean sources). Both KETO groups will aim to achieve < 10% of energy intake from carbohydrates, 20–30% of energy from protein, and 60–70% of energy from fats. Each KETO group will include the specified ingredients as their primary fat source (i.e., canola oil in the KETO-Can group and coconut oil and butter in the KETO-Sat group). A daily multivitamin supplement will be provided to all participants, regardless of diet, to avoid potential nutrient deficiencies. Each month, as needed, participants will be supplied with ingredients for their respective diets to promote adherence to key nutrients. Ingredients are sourced from grocery stores and/or food service distributors.

All participants will receive nutrition counselling by a nutrition expert (i.e. an RD or person with a degree in Nutrition with relevant clinical experience) from the study team either in person or remotely (based on participant’s preference and availability). These nutrition experts will provide counselling to participants across all three study groups. Nutrition counselling for participants living with T2D will be provided by RDs with Diabetes Educator certification. Initial nutrition education sessions will be completed at baseline and two weeks with a detailed overview of diet requirements and related disease management as required. Follow-up sessions will then be held monthly for the duration of the study (either via videoconferencing, telephone, or in person depending on preference), with a phone call check-in every two weeks in between monthly visits to support dietary adherence (Table 1).

Table 1.

Nutritional counselling outline of content reviewed and timeline

Timepoint	Duration	Content	Delivery method
Baseline 1 st consultation	1–1.5 h	Explanation of prediabetes/T2D, glucose management and ketosis (if in keto group) Explanation of the assigned diet Provide recipe booklet and handouts	In person/Videoconference/Telephone
2 weeks 2nd consultation	30–40 min	Explanation of food serving/portion sizes Menu planning activities Review 24-h recall	In person/Videoconference/Telephone
1-month 3rd consultation	30 min	Review 24-h recall Individualized follow-up Explanation of food labels Food label reading activities	In person/Videoconference/Telephone
1.5 months Check-in	15 min	Midpoint check-in – reminder of next visits	Telephone
2 months 4th consultation	30 min	Review 24-h recall Individualized follow-up	In person/Videoconference/Telephone
2.5 months Check-in	15 min	Midpoint check-in – reminder of next visits	Telephone
3 months 5th consultation	30 min	Review 24-h recall Individualized follow-up	In person/Videoconference/Telephone
3.5 months Check-in	15 min	Midpoint check-in – reminder of next visits	Telephone
4 months 6th consultation	30 min	Review 24-h recall Individualized follow-up	In person/Videoconference/Telephone
4.5 months Check-in	15 min	Midpoint check-in – reminder of next visits	Telephone
5 months 7th consultation	30 min	Review 24-h recall Individualized follow-up	In person/Videoconference/Telephone
5.5 months Check-in	15 min	Midpoint check-in – reminder of next visits	Telephone
6 months 8th and final consultation	30 min	Review 24-h recalls Review 6 month bloodwork Review anthropometric/body composition results Provide final recommendations on healthy eating patterns, maintaining stable body weight, and transitioning away from keto diet (if applicable)	In person/Videoconference/Telephone

Criteria for discontinuing or modifying allocated interventions {11b}

There are no pre-specified criteria for stopping the study due to safety concerns. Withdrawal from the study will be considered if a participant develops a condition that falls under the exclusion criteria or a medical condition that may warrant exclusion from further participation in the trial. The Principal Investigator (PI) will evaluate the condition in consultation with the study physician and the study team. Examples of relevant clinical presentations that would prompt further investigation include apparent allergies or sensitivities to food products used in the study, hypoglycemia, hypotension, metabolic acidosis, and hypercalciuria with associated nephrolithiasis [4, 26]. Withdrawal will be considered if the condition poses significant risk to the participant’s health or compromises the integrity of the study results. The study physician may elect to modify medications in order to allow the participant to continue in the study. Participants will also be withdrawn if they notify any member of the study team of unwillingness to continue or are lost to follow-up. Participants who fail to respond to two written emails (first contact, and a second follow-up) and a follow-up phone call will be withdrawn. Withdrawn participants will be included in the intention-to-treat analysis of the primary endpoints, if consent is provided at the time of withdrawal.

Strategies to improve adherence to interventions {11c}

Provisions to participants that may support adherence include supplementary staple foods for the study diets (canola oil, butter, coconut oil, whole grain products, etc.), and an honorarium in the form of a $100 grocery store gift card. Individualized dietary counselling will also be an appealing factor for participants who aim to lose weight. Reimbursement for parking and public transportation expenses will be provided.

The participants’ regular visits to the HNRU plus phone calls will allow the RD to provide support and motivate participants to obtain maximum adherence to assigned diets. Recipe booklets tailored to each diet with inclusion of primary ingredients will be provided to facilitate dietary adherence.

Relevant concomitant care permitted or prohibited during the trial {11d}

All participants will be encouraged to maintain their usual physical activity habits throughout the entire intervention and limit alcohol consumption based on Canada’s Guidance on Alcohol and Health. Fluctuations in female hormones influence metabolic variables [27–29]; thus, women will be tested during the follicular phase of their menstrual cycle (days 3 to 9) whenever a menstrual cycle is present [30]. Participants should continue their regular medications, which will be recorded, as will changes in medications.

Provisions for post-trial care {30}

We do not anticipate harm arising from the trial. Thus, post-trial care and compensation are not applicable.

Outcomes {12}

General procedures and reporting of outcomes

Participants will attend three formal in-person assessments for data collection at baseline, three and six months, each scheduled on 2 consecutive days (Table 2). Participants experiencing fever, cough, runny nose or gastrointestinal symptoms will be rescheduled, as active infections can confound CRP measurement.

Table 2.

Expected timeline for participants enrolled in the study. Activities during study visits at baseline, 3 months (3m), and 6 months (6m) are distributed across two days.

Unless otherwise specified, outcomes will be expressed as mean change in values with confidence intervals from baseline to three months and six months timepoints (Table 2). The rationale for the primary, secondary and exploratory variables is as follows:

Primary outcomes

TG (mean change from baseline, mmol/L)

TG is a key outcome measurement in trials involving KETO diets because TG metabolism is expected to be altered in the state of ketosis. Reduced circulating insulin concentrations during ketosis are predicted to result in decreased TG synthesis, and increased lipolysis with ketogenesis leading to increased TG removal from blood [31]. Previous studies of KETO diet interventions confirm this effect, showing consistent decreases in circulating TG on a LCD independent of the fat source [4, 31, 32]. Thus, the change in TG concentrations can be used to evaluate the effectiveness of treatment.

LDL-cholesterol (mean change from baseline, mmol/L)

Research demonstrates a causal relationship between LDL-C and atherosclerotic CVD, with LDL-C used to measure thresholds and targets for lipid-lowering therapies [33]. Therefore, monitoring LDL-C will provide insight into the safety of the diets studied and therapeutic comparison to current lipid-lowering medications. The metabolism of LDL-C while on the KETO diet is quite complex; although concentrations might expect to be elevated with increased consumption of SFA, accompanying lower circulating insulin inhibits HMG-CoA reductase (involved in cholesterol synthesis) and may subsequently decrease or stabilize LDL-C concentrations [4, 31]. LDL-C concentrations are expected to be lower in the KETO-Can treatment arm, and concentrations in the KETO-Sat arm are expected to be similar if not greater than the control arm.

Secondary outcomes

Blood glucose (mean change from baseline, mmol/L)

Fasting blood glucose is included as an outcome to assess the effects of diet on diabetes risk or T2D management. Following a KETO diet has been shown to lower fasting blood glucose [34, 35]. This is thought to be secondary to reduced hepatic gluconeogenesis [36]. However, glucose concentrations are still expected to be kept within normal range secondary to gluconeogenesis from amino acids (earlier on) or glycerol (later on) [37]. Thus, fasting blood glucose concentrations are predicted to be lower in both treatment groups compared with the control group at the study’s conclusion.

Plasma insulin (mean change from baseline, pmol/L)

Measuring insulin concentrations will help assess the KETO diet’s impact on diabetes risk or T2D management. Individuals with T2D often have elevated circulating insulin due to insulin resistance [38]. The KETO diet is associated with lower insulin secretion secondary to reduced requirement [34, 39]. Consequently, insulin concentrations should be decreased in the treatment groups on the KETO diets with reduced carbohydrate consumption compared with the control group.

HbA1c (mean change from baseline, %)

HbA1c is a marker used by healthcare professionals to assess longer-term control of diabetes risk or T2D management (over the previous three months). The KETO diet has consistently shown greater decreases in HbA1c compared with diets higher in carbohydrates [34, 40, 41]. Measurement of HbA1c will directly assess the influence of diet on diabetes risk or T2D management, and a greater decrease in HbA1c is expected in the treatment groups compared with the control group.

Apolipoprotein B100 (ApoB100) (mean change from baseline, mmol/L).

ApoB100 concentrations more accurately reflect the number of atherogenic lipoproteins compared to LDL-C alone and better predict cardiovascular event risk [33]. Measuring ApoB100 will provide a more comprehensive assessment of lipid metabolism, especially if TG are high or when HDL-C is low, which may occur in the initial stages of the KETO diet. Considering the more recent introduction of ApoB100 to the research literature compared with LDL-C, few studies have examined the impact of diet on ApoB100 concentrations or other more atherogenic particles, such as chylomicron remnants. A recent meta-analysis suggests an increase in ApoB100 levels with the KETO diet [42] but these studies did not control for type of fat intake. As the KETO-Sat group will have higher intake of SFA compared to the KETO-Can and control groups, a greater increase in ApoB100 concentrations is predicted in the KETO-Sat group compared with the other groups.

Inflammation

CRP (mean change from baseline, mg/L) is a marker of systemic inflammation, thus its measurement will reflect the inflammatory status associated with each diet [43]. CRP is an independent CVD risk factor. Since CRP can increase rapidly due to infection, measurement will be done on two consecutive days to rule out ongoing infection. Because PUFA have been shown to reduce inflammation, it is expected that the KETO-Can group will have the lowest concentrations of CRP [22].

Immune cell phenotype and function

As increased body weight, inflammation and insulin resistance are associated with immune dysfunction [17], changes in markers of the immune system will be reflective of the impacts of each diet on weight loss, glucose metabolism and systemic inflammation. Immune cell phenotypes will be characterized using conventional flow cytometry to monitor changes in cell populations and their activation/inhibition markers. Proliferation and ex vivo cytokine production immune assays will be conducted to measure immune cell function. Intracellular staining post stimulation will be used to measure which cell types produced key cytokines using conventional flow cytometry. Immune variables will be reported as the mean absolute values and as the mean % change from baseline at three and six months. Immune cells measured include T cells (CD3, CD4 +, CD8 +, CD45RA +, CD45RO +), Tregs (FOXP3 +), B cells (CD20), macrophages (CD14), Natural Killer (NK) cells (CD56 +), and activation markers (CD11b +, CD25 +, CD28 +, CD86 +). Cytokines measured include interleukin (IL)−1β, IL-2, IL-6, IL-10, interferon gamma (IFN)-γ, and tumour necrosis factor alpha (TNF)-α.

Exploratory outcomes

Adherence to dietary interventions

Adherence is a key factor to assess during a nutritional study to help ensure measured outcomes can truly be associated with the manipulated variables. A total of three 24-h recalls will be collected at baseline on two weekdays and one weekend day to assess dietary intake prior to initiating the allocated diet. Adherence to the assigned dietary intervention will be measured using a validated, internet-based 24-h recall questionnaire (ASA-24®) [44]. A total of eleven 24-h recalls will be collected throughout the study at scheduled follow-ups: three at three and six months, and one each at two weeks, one month, two months, four months, and five months (Table 2). Overall adherence from two weeks to six months will be reported as an exploratory outcome. From these data, energy intake (kcal), macronutrient intake (g) and distribution (% of energy intake), SFA (g and % of energy), MUFA (g and % of energy), PUFA (g and % of energy), and fibre intake (g) will be calculated at monthly intervals. Change from baseline at three and six months will also be reported. Selected vitamins and minerals will be reported by weight (mg or mcg).

For adherence, participants will be classified as highly compliant if eight out of 11 (or > 70%) recalls meet dietary macronutrient distribution objectives; moderately compliant if five-seven out of 11 (45%−69%) recalls meet the objectives; and low adherence if four or fewer (< 45%) recalls meet the objectives.

Measurement of fatty acids (total (mmol/L) and individual fatty acids (proportion of total)) will provide supporting information on participant adherence to the assigned dietary intervention, as each diet is expected to be associated with alterations in total plasma fatty acids and red blood cell (RBC) membrane phospholipids as a measurement of acute dietary intake (i.e. total fatty acids) and long-term adherence (i.e. RBC reflecting the last three months).

Blood ketones (mmol/L) will be measured and reported as absolute values at baseline and monthly thereafter until the six-month completion mark in the KETO treatment groups (Table 2), as an objective measure of higher fat intake, using a FreeStyle Libre 2 monitor with FreeStyle B-Ketone testing strips (Abbott, Montreal, QC, Canada). Participants will be considered in ketosis with blood ketone concentrations at or above 0.5 mmol/L.

Additional assessments

Anthropometric measurements and body composition

Weight, waist, and hip circumference will be measured (cm) at time of screening, baseline, and monthly thereafter until the six-month completion mark (Table 2). Height (cm, measured only at screening) and weight (kg) will be used to calculate body mass index (BMI, kg/m²). Values will be reported as changes from baseline. Body composition analysis will be done at baseline, three and six months and fat and lean mass will be reported as changes from baseline (kg and % body weight).

Anthropometric outcomes will be measured in duplicate including height (SECA 264 Stadiometer X, Hamburg, Germany) and weight using digital scales (Health o meter® Professional Remote Display, Sunbeam Products, FL, USA), waist and hip circumferences. Participants will remove shoes and wear light clothing. The mean of two measurements will be used. Body composition will be assessed using bioelectrical impedance analysis (InBody 770 BIA, InBody Canada, Ottawa, ON, Canada) and includes weight, body fat percentage, skeletal muscle mass, and body fat mass.

Complete blood count (CBC) with differential

CBC will be reported as absolute values at baseline, three and six months by Alberta Precision Laboratories and used to complement our immune data as well as to assess if the individual has an infection that could confound the CRP values. Variables include white blood cells (10⁹/L), RBC (10¹²/L), hemoglobin (g/L), mean corpuscular volume (fL), hematocrit (L/L), mean corpuscular hemoglobin concentration (g/L), red cell distribution width (%), platelets (10⁹/L), with differentiation of white blood cell types concentrations (neutrophils/monocytes/lymphocytes/eosinophils/basophils/immature granulocytes/nucleated RBC).

Blood pressure

The KETO diet has been shown to reduce both systolic and diastolic blood pressure in previous studies, although with mixed results in participants with T2D [4], which may reduce CVD risk. Systolic and diastolic blood pressure (mmHg) will be measured at time of screening, baseline, three and six months. Blood pressure will be measured in duplicate using an automated device (Welch Allyn, Baxter, Mississauga, ON, Canada), in seated participants, with one minute between measurements. The mean of the two results will be used. If the first two systolic readings differ by more than 10 mm Hg, an additional reading will be obtained, and the mean of the two closest results will be used.

Appetite

Appetite awareness and satiety cues play a role in managing oral intake while adhering to a specified diet. Given the known impact of both the KETO diet [4] and increased fibre intake on appetite regulation, it will be relevant to the study to collect data. Appetite will be measured using a validated questionnaire with a visual analogue scale [45] at baseline, three and six months and reported as the absolute value. This questionnaire measures hunger, satisfaction and fullness on a linear scale using subjective answers provided by the participants before and after meals. Six questionnaires are collected for one day (three meals) at each of the three timepoints.

GI symptoms

A gastrointestinal tolerance questionnaire will be completed at baseline, three and six months to assess whether any of the diets is causing systematic changes in gastrointestinal symptoms and reported as absolute values. This questionnaire assesses gastrointestinal symptoms using an ordinal scale for severity, including pain, distension, gas, flatulence, as well as stool consistency (based on Bristol stool ratings) and frequency of bowel movements.

Physical activity

Physical activity will be measured at baseline, three and six months and reported as absolute values. Physical activity will be measured using the International Physical Activity Questionnaire to assess minutes/hours of vigorous/moderate/walking and sitting activities in the most recent seven days. [46] Step counts/day will be measured using a digital pedometer (Piezotype, StepsCount, Ottawa, ON, Canada). Sealed pedometers will be provided to participants at their baseline and three-month assessment visit, and provided at the five-month visit for use the week prior to returning for their final six-month visit at study completion. They are asked to wear the device during waking hours for three days, including one weekend day and two weekdays. The mean of the three days of physical activity (steps/day) will be recorded.

Background and demographic information

A questionnaire will be administered at screening (Table 2) to capture the following information: Sex assigned at birth (male/female), gender (man/woman/non-binary/gender-fluid/trans man/trans woman/two-spirit/doesn’t identify with the options provided/prefer not to say), age (years), ethnic origin (Caucasian (white)/Black/Indigenous (aboriginal)/South Asian/West Asian/Southeast Asian/Japanese/Arab/Latin American/Chinese/Korean/prefer not to answer), weight stability (< 3% change in 6 months), education (highest level completed: high school/some college (no degree)/professional degree/bachelor’s degree/graduate degree/prefer not to answer), marital status (single (never married), married or domestic partnership/widowed/divorced/separated/prefer not to answer), employment status (employed > 30 h weekly/employed < 30 h weekly/not employed (looking/not looking for work)/student/homemaker/prefer not to answer), household income (ranges from 0-$25,000 in $25,000 increments to $200,000 and up, or prefer not to answer), medical history (includes major diagnoses of diabetes (type 1 and 2)/CVD/hypertension/hypercholesterolemia/liver or kidney disease/cancer/pancreatitis/eating disorder/anemia/COVID-19/other), current medications and supplement use.

Quality of life

Throughout the study, we expect to observe changes in the physical and mental health status of our participants as other health-related outcomes are impacted (i.e. weight, glucose and lipid measures). Information on basic mental and physical health and activity limitations will be obtained at baseline, three and six months using the Centers for Disease Control and Prevention Health Related Quality of Life Healthy Days Measure (CDC HRQOL-14) [47]. This questionnaire assesses perceived mental and physical health challenges in the past thirty days and provides a score, which will reported as an absolute value for each timepoint.

Ingredient consumption

Participants will report monthly on their consumption of the study-provided foods (i.e. canola oil (mL) for KETO-Can, butter (g) and coconut oil (mL) for KETO-Sat, oatmeal, brown rice, whole-wheat pasta (g/cups) for LFD) (Table 2). Capturing this data will support dietary adherence analysis and the contribution of specific ingredient components on trial outcomes and will be reported as absolute values.

Current medications and supplements

Participants will provide a list of current medications and supplements and report any changes in health. Changes in lipid-, glucose- or blood pressure-lowering medications will be noted as a potential confounder.

Additional lipid markers (included in the lipid panel)

Total cholesterol (mmol/L), HDL-C (mmol/L) and non-HDL-cholesterol (mmol/L) will be measured at baseline, three and six months by Alberta Precision Laboratories. Total cholesterol and HDL-C are required to calculate LDL-C. These measures will serve as additional co-factors for cardiovascular risk assessment.

Optional microbiome sub-study

Participants may opt-in or opt-out of this portion of the study. For those consenting to this sub-study, fecal samples will be collected by participants at home at baseline, three and six months. A collection device (FecesCatcher, TAG HEMI) and a fecal collection tube (DNA/RNA Shield™, Zymo Research Corp, United States) will be provided in a sealed plastic bag along with the instructions for proper handling of samples. Fecal samples will be kept at room temperature until brought to the HNRU where they will be stored at −80 °C for subsequent analysis.

Participant timeline {13}

From the study’s start date of Dec 12, 2022, until enough participants have been enrolled (175 total participants; expected stop date July 31, 2026), participants will be enrolled in the trial on a rolling basis. Pre-screening and recruitment for the trial began on July 1, 2023 with the first participant enrolled in the study on August 14, 2023. Respondents are assessed for eligibility in a two-step process; they first complete an online survey, known as “pre-screening” for basic eligibility, and based on this questionnaire, they are either declined (i.e., they are ineligible), or they are invited for a screening visit at the HNRU where eligibility is confirmed via anthropometric data and serum lab values (Table 2). Information collected at screening and baseline is reviewed by the study physician to confirm each participant's fitness to be included in the trial. Participants are enrolled in the study at their baseline visit. The timeline for an individual’s expected participation in the study from the start of screening to the six-month mark (endpoint for participants) is outlined in Table 1. Note that the baseline, three and six month visits require visits to the study site on two consecutive days, with baseline measurements completed prior to diet allocation (Table 2).

Food intake will be assessed with 24-h recall questionnaires, as outlined previously. Details regarding nutritional counselling provided during the study is further outlined in Table 1.

All nutrition counselling (Table 1) will be conducted either in person or via videoconferencing/telephone based on participant preference. Baseline nutrition counselling will be specific to the arm of the study and participants will be provided with a recipe booklet and handouts tailored to their diet allocation during the baseline week. For the three-month nutrition counselling, specific to the arm of the study, the counsellor will review 24-h recalls, three-month blood work, and anthropometric/body composition results with the participant to provide recommendations as needed. At month one, two, four and five, the counsellor will review the 24-h recalls and provide recommendations to enhance adherence as needed For the final session at six months the counsellor will review 24-h recalls, six-month blood work, and anthropometric/body composition results to provide final recommendations based on participant preferences (e.g. to remain on the allocated diet or transition to another dietary pattern) and Canada's Food Guide (healthy eating pattern), for maintaining a stable body weight, and transitioning safely away from the KETO diet (if applicable). Participants are also provided with resources to access ongoing nutrition support, if desired, following study completion. The session will be either in person or remotely during the six-month follow-up week. At study completion, a letter containing a table of results is reviewed with the participants detailing lab values and anthropometrics, outlining changes from baseline to 3 months to 6 months on the study. Participants can obtain a copy of this results letter, if they choose (Table 2).

Sample size {14}

A comparison of dietary interventions needs to consider how the diet composition (e.g. SFA, total fat, total carbohydrate and fiber) affects outcome measures. The two main comparisons will be between the control LFD and the two KETO groups. For TG (and taking into account sequential measurements at three and six months), a power calculation indicates that n = 45 per group will be sufficient to detect a statistically significant difference with a standard deviation of 0.60 mmol/L, a power of 0.8, and alpha of 0.05, estimating a correlation between timepoints of 0.6. A SD for TG of 0.6 mmol/L was derived from a previous study comparing the effects of replacing dietary SFA with UFA on plasma lipids in participants [9].

When comparing the two KETO arms, the replacement effect of the fatty acid profile is important. Replacing SFA with MUFA led to a reduction of 10–12% in LDL-C and was statistically significant with n = 42 per group [9]. Thus, we should also have sufficient power to detect a difference in LDL-C between the KETO groups. The correlation between timepoints was estimated to be 0.8.

Based on previous studies from our group [48, 49], an n = 45 per group will also provide enough power to assess significant differences in secondary outcomes including weight, systemic inflammation and immune function. Assuming an attrition rate of 30% during the intervention period, we will aim to recruit 175 participants.

Recruitment {15}

Participants will be recruited from the city of Edmonton and surrounding areas using a variety of approaches, including physical spaces (research posters, pamphlets), the media (social media, newspaper, radio, TV), University communications, obesity clinics in the Edmonton area (physician referrals), word of mouth, and research lists (names of participants who previously gave consent to be contacted for future studies). The study will also be listed on the websites of HNRU, ADI and Be The Cure websites and a specific study website [50].

Assignment of interventions: allocation

Sequence generation {16a}

Participants will be randomized to their assigned intervention using a computer-generated blocked randomization process to ensure a balanced 1:1:1 allocation of participants into three groups. Randomization will be completed following the first baseline visit (day 1).

Concealment mechanism {16b}

The allocation will be revealed to the participant by the research coordinator on day two of the baseline visit.

Implementation {16c}

The randomization will be done by a statistician not involved in the conduct of the trial. The research coordinator, who is not privy to the generated block-randomization details (e.g. block size), will enroll participants and assign them to interventions.

Assignment of interventions: Blinding

Who will be blinded {17a}

This is an open-label trial because participants are required to know which diet to follow, study staff must appropriately allocate supplementary foods, and nutrition experts need to counsel participants based on their diet assignment. Thus, blinding of research staff and participants is not possible. However, a technician and statistician blinded to study groups will perform all analyses.

Procedure for unblinding if needed {17b}

Not applicable because the study coordinator, nutrition counsellors and participants are all aware of the allocated intervention.

Data collection and management

Plans for assessment and collection of outcomes {18a}

All study team personnel will be trained and follow standard operating procedures for use of all equipment during study visits, including blood pressure machines and point of care devices, as well as for taking anthropometric measurements.

Fasting blood collection

Blood samples will be collected from participants after a 9–12 h overnight fast. Blood will be collected into BD Vacutainer® tubes (Becton Dickinson, Franklin Lakes, NJ, USA) with spray coated lithium heparin and gel or spray-coated with dipotassium ethylenediaminetetraacetic acid (K2 EDTA) for plasma separation, and serum separating tubes (SST) for serum separation. Serum vacutainers will be kept at room temperature (RT) for 15–30 min to allow for clot formation and then stored at 4 °C until processed.

Analysis of clinical variables

At the screening visit, blood samples to measure HbA1c will be sent to Alberta Precision Laboratories. At baseline, three, and six months blood will be collected on two consecutive days. On day one, Alberta Precision Laboratories will analyze samples for TG, total cholesterol, HDL-C (enzyme colorimetric assays), fasting glucose (UV testing using an enzymatic reference method with hexokinase), CRP (immunoturbidimetric assay) all using an automated photometric analyzer (Roche Cobas c503). Insulin will be measured with an electrochemiluminescence immunoassay (Roche Cobas e801). The remaining blood and plasma/serum will be collected and stored for further analysis. On day two, the collected blood sample will be analyzed by Alberta Precision Laboratories for CBC with differential (CBC, via whole blood fluorescent flow cytometry (Sysmex XN10), HbA1c with a turbidimetric inhibition immunoassay for hemolyzed whole blood (Roche Cobas c513) and CRP (immunoturbidimetric assay using the Roche Cobas c503) to rule out any ongoing infection, i.e., CRP > 10 mg/L. These data will be accessed and reviewed through the electronic medical record. LDL-C will be calculated using the following equation: Total cholesterol $-$ HDL-C $-$ (TG/2.2). Non-HDL-C will be calculated as total cholesterol $-$ HDL-C. The Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) index will be calculated using the equation: fasting insulin (μU/L) × fasting glucose (nmol/L)/22.5 [51].

Whole blood processing

In the Richard lab, at the University of Alberta, K2 EDTA tubes and SST serum tubes will be centrifuged at 1350 × g for 10 min at 22 °C. Plasma and serum will be aliquoted for storage at −80 °C for subsequent analysis. After plasma separation, the remaining buffy coat and erythrocytes are resuspended with 3 mL of 1% bovine serum albumin (BSA, Sigma-Aldrich, Co., St. Louis, MO, USA) in phosphate-buffered saline (PBS) pH: 7.2–7.4 containing diH₂0, NaCl (0.14 M), NaH₂PO₄ (9.67 mM), KH₂PO₄ (1.47 mM), KCl (2.68 mM), Fisher Scientific) and layered onto Ficoll-Paque (HISTOPAQUE ® −1077, Sigma-Aldrich), then centrifuged (1558 × g, 30 min with no brake, 20 °C) for peripheral blood mononuclear cells (PBMC) isolation using density centrifugation. The PBMC bands at the gradient interface of 1% BSA in PBS and Ficoll-Paque are transferred to a 50 mL conical tube, brought to 40 mL with 1% BSA in PBS, and centrifuged (478 × g, 5 min, 4 °C to pellet PBMCs. Supernatant is discarded and PBMCs are then washed with 10 mL 1% BSA in PBS, and centrifuged as previously. PBMCs are then resuspended in 5 mL of 5% fetal calf serum (FCS) in complete culture media (CCM) containing Roswell Park Memorial Institute 1,640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 25 mmol/L 4-(2-hydroxyethyl)−1-piperazineethanesulfonic acid (HEPES, Corning, Manassas, VA, USA), 2.5 μmol/L 2-mercaptoethanol (Thermo Fisher Scientific, Grand Island, NY, USA), and 1% (v/v) antibiotic/antimycotic solution (AB/AM, Sigma-Aldrich) (5% CCM), and an aliquot is mixed 1:1 with trypan blue (diluted 1:1 with PBS, Corning) to count cells using a hemocytometer and the trypan blue membrane dye exclusion method. After mitogen assay setup (described in Ex-vivo mitogen stimulation of PBMCs), a 1 mL aliquot is transferred to microtubes, and centrifuged (906 × g, 2 min, 10 °C). After discarding the supernatant, the pellet is washed with 500 μL sterile PBS, centrifuged as above, and the supernatant discarded. The pellet is then stored at −80 °C for subsequent analysis. The remaining cell suspension is then centrifuged (478 × g, 5 min, 4 °C) and the supernatant discarded. PBMCs are resuspended in 3 mL of 20% CCM containing 10% (v/v) dimethyl sulfoxide (DMSO, MP Biomedicals, Solon, OH, USA) added before use. The cells are aliquoted in three 1 mL cryovials and immediately frozen at −80 °C using a freezing container (Mr. Frosty™ Cryo 1 °C, Nalgene ®, Rochester, NY, USA) then transferred to liquid nitrogen the next day for long-term storage.

The RBC fraction of the histopaque gradient is washed with 5 mL 0.9% saline and centrifuged (453 × g, 5 min, 4 °C). The supernatant is discarded and the RBCs washed and centrifuged as previously. RBCs are lysed by adjusting to 3 mL with deionized water and mixing with a 1 mL aliquot stored at −80 °C for subsequent lipid analysis.

Immune Phenotype and Function

To conduct the analysis, immune cell subsets from fresh whole blood will be identified by direct immunofluorescence assay developed by our group [49]. Briefly, 96-well V-bottom plates (Costar ®, Kennebunk, ME, USA) are pre-conditioned with 200 μL 5% (v/v) FCS (Thermo Fisher Scientific, Waltham, MA, USA) in PBS with AB/AM (IF buffer) for at least 30 min before discarding and adding 100 μL of whole blood. The RBCs are lysed using 200 μL of 1X RBC lysis buffer (BioLegend, San Diego, CA, USA) as follows: After a 10–15 min incubation at RT, the plate is centrifuged (428 × g, 2 min, 10 °C) to pellet immune cells. The supernatant is needle aspirated, and RBC are lysed for 5–10 min, and plate centrifuged as before. If RBCs are still evident, a third lysis step (5–10 min) is performed. After RBC lysis, immune cells are washed twice with 200 μL IF buffer and incubated with 80 μL of Zombie UV fixable viability dye (BioLegend) diluted to 1:100 in PBS for 15 min at RT. Following the incubation, 20 μL of antibody cocktails (panels 1–5, excluding FOXP3) are added for 30–60 min at 4 °C in the dark. The antibody conjugates, staining reagents, and the corresponding multicolour flow cytometry panels are designed to identify immune cell subsets and activation markers (Panel 1—T helper cell, Panel 2—T cell, Panel 3 – regulatory T cells (T reg), Panel 4—B cell, Panel 5—Monocyte, Dendritic and NK cells) and are shown in Supplementary Table S1. After incubation cells are washed with IF buffer twice, as previously, they are resuspended in 200uL of 1% paraformaldehyde in PBS for 1 h to fix the cells.

After fixation, cells stained with panels 1, 2, 4, and 5 are transferred to fluorescence-activated cell sorting (FACS) tubes containing 100 μL IF buffer and stored in the dark at 4 °C until flow cytometry is performed. T reg cells (panel 3) are centrifuged (428 × g, 2 min, 10 °C) and supernatants aspirated. Then, T reg cells are resuspended in 200uL of 1 mg/mL saponin (Sigma Aldrich, Oakville, ON, Canada) diluted in IF buffer (saponin buffer) and incubated in the dark on ice for 5–10 min. Cells are then centrifuged as above and incubated with 20 μL of FoxP3 diluted in saponin buffer in the dark for 30 min on ice followed by 20 min at RT. After washing twice with 200 μL saponin buffer and twice with 200 μL IF buffer as before, cells are resuspended in 200 μL of IF buffer and transferred to FACS tubes containing 100 μL of IF buffer.

Compensation beads (AbC™ Total Antibody Compensation Bead Kit, Thermo Fisher Scientific) are used to control for fluorochrome spillover. Tandem-specific compensations are employed to account for differences in compensation lot to lot and vial to vial. Briefly, microtubes and FACS tube are labelled for each antibody (n = 5). Antibodies are diluted 1:100 (BV421, BV510, APC, FITC, AF647, PerCP, CD74/HLA-DR BV711, BV605, AF700) or 1:200 (CD185 PE-Cy7, CD45RA PE-Cy7, CD127 APC-Cy7, CD45RO BV711, CD40 PE-Cy7, CD10 BV711, CD273 APC-Cy7, CD86 PE-Cy7, IgM APC-Cy7, IL-13 PE-Cy7, IL-10 PE-Dazzle594, IL-6 PE-Cy7, CD196/CCR6 BV711) or 1:300 (PE) using IF buffer. One drop of positive and negative beads are added to FACS tubes. Previously diluted antibodies (50 μL) are then mixed with the beads and incubated at 4 °C in the dark for 30 min at 4 °C. Beads are then washed twice with 200 μL of IF buffer with centrifugation (428 × g, 2 min, 4 °C), discarding the supernatant. Compensation beads are resuspended in 300–600 μL IF buffer and stored at 4 °C in the dark. All samples, including compensation beads, are acquired within 72 h using a BD LSR-Fortessa X-20 flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed according to the relative fluorescence intensity using a platform for single-cell flow cytometry analysis (FlowJo 10.8.1, Becton Dickinson).

The flow cytometer machine goes through rigorous quality control, including periodic calibration, and is located at the Faculty of Medicine and Dentistry Flow Core at the University of Alberta, which is a recognized laboratory by the International Society for Advancement of Cytometry. Quality control of acquired samples will also be monitored using a method that adopts algorithms for the detection of anomalous data (flowAI) as previously described [16, 49]. Data are processed automatically, with the call of an R function, by optimizing flow rate, signal acquisition, and dynamic range [52].

PBMCs will be the starting point gate of all immune subsets, which will be determined using established gates based on morphological characteristics of forward and side scatter. Doublets are then removed using size exclusion, and viable cells are gated using Zombie UV fixable viability dye (Biolegend) to ensure identified populations are accurate. Fluorescence minus one (FMO) controls will be used to establish positive staining when needed. All analyses were carried out by one individual and gates reviewed by another researcher to minimize interobserver variability.

Ex-vivo mitogen stimulation of PBMCs

The quantification of cytokine secretion by PMBCs stimulated with mitogens is used to evaluate the effector function of immune cells. Briefly, PBMCs are cultured at 1.00 × 10⁶ cells/mL in 3 mL of CCM supplemented with 10% FCS. The PBMCs are cultured without mitogen (control) or stimulated with one of: phytohemagglutinin (PHA, 5 mg/mL; Sigma-Aldrich), lipopolysaccharide (LPS, 5 μg/mL, Thermo Fisher Scientific, Carlsbad, CA, USA); Phytolacca americana (pokeweed, PWM, 55 μg/mL) mitogens. Culture tubes are incubated at 37 °C with 5% CO₂ in a cell culture incubator. Brefeldin A (BioLegend) is then added at 1X concentration to PHA and LPS tubes and all mitogens incubated for 4 h. Mitogen tubes are then centrifuged (428 × g, 5 min, 4 °C) to pellet PBMCs. The supernatant is aliquoted and frozen at −80 °C for cytokine quantification. PHA and LPS cell pellets are resuspended with 150 uL IF buffer and transferred to pre-conditioned 96-well V bottom plates for intracellular staining (described elsewhere). PWM and control cell pellets are resuspended in 500 μL PBS, transferred to microtubes, and centrifuged (906 × g, 2 min, 10 °C). After discarding the supernatant, the cell pellet is stored at −80 °C for subsequent analysis.

Intracellular staining of mitogen stimulated PBMCs

PHA and LPS pellets previously transferred are centrifuged (428 × g, 5 min, 4 °C) and the supernatant aspirated. The cells are washed with 200 μL of IF buffer as before. The cells are then stained with 80 μL of Zombie UV fixable viability dye diluted to 1:100 in PBS for 15 min at RT. Then, 20 μL of antibody cocktail is added to characterize immune cell lineage (panels 6 and 7, Supplementary Table S1) for 30 min at 4 °C in the dark.

Next PBMCs are washed twice with IF buffer then resuspended in 200 μL of 1X Nuclear Fix solution (Transcription factor buffer, BioLegend), incubated at RT in the dark for 45–60 min, then centrifuged (428 × g, 2 min, 10 °C) and the supernatant aspirated. The cells are then washed three times with 200 μl of 1X perm buffer. Cells are incubated with 20 μL of intracellular antibody cocktail diluted in perm buffer (Panel 6b and 7b, Supplementary Table 1) for 30 min at RT in the dark, then washed three times with perm buffer and twice with IF buffer before resuspending in 200 μL IF buffer and transferring to FACS tubes containing 100 μL IF buffer. All samples are acquired within 72 h using a BD-LSR Fortessa X-20 conventional cytometer as described previously.

Quantification of ex-vivo cytokine secretion

Concentrations of interleukin (IL)−1β, IL-2, IL-6, and IL-10, IFN-γ, and TNF-α in the supernatant are determined in duplicate by commercial ELISA kits (DuoSet ®, R&D Systems, Minneapolis, MN, USA) according to the instructions of the manufacturer. The detection limits for the cytokines are as follows: IL-1β (250–3.91 pg/mL), IL-2 (1000–15.6 pg/mL), IL-6 (600–9.38 pg/mL), IL-10 (2000–31.3 pg/mL), IFN-γ (600–9.38 pg/mL), and TNF-α (1000–15.6 pg/mL). Cytokine concentrations are quantified using a microplate reader with an intra-assay coefficient of variation < 10% and R² of the standard curve ≥ 0.99. Samples above high standard were diluted onto the linear portion of the standard curve.

Immune cell recovery

PBMC previously stored in liquid nitrogen are rapidly thawed in a 37 °C water bath for 5 min, then transferred to a 9 mL pre-warmed 37 °C aliquot of 10% CCM. PBMCs are centrifuged (453 × g, 5 min, 21 °C. After discarding the supernatant, cells are rinsed with 10 mL of prewarmed 10% CCM to remove DMSO. Following another wash with centrifugation, PBMCs are resuspended in 5 mL of pre-warmed 10% CCM and transferred to 12 mL cell culture tubes to rest overnight in an incubator at 37 °C with 5% CO₂. Before use in immune assays, PBMCs are centrifuged as above and resuspended in 2 mL 10% CCM and lymphocytes counted as described earlier. All cell counts are carried out by the same person to minimize inter-observer variation.

Proliferation assay

The proliferation of T cells is measured using alamarBlue ® fluorescence (BUF012A, Bio-Rad Laboratories, Hercules, CA, USA). PBMCs are either stimulated with anti-CD3 and anti-CD28 (BioLegend, as above) or unstimulated. Briefly, 48 wells of black flat bottom 96-well cell culture plates (NUNC ®, Thermo Fisher Scientific, Roskilde, Copenhagen, Denmark) are coated with 100 μL of 5 μg/mL anti-CD3 stock solution, and 48 wells are coated with sterile PBS, then incubated overnight at 4 °C. Then all wells are needle aspirated and washed twice with 200 μL sterile PBS. For the unstimulated and stimulated wells, 200 μL and 196 μL of 10% CCM is added, respectively. For this assay, the same number of cells is added to each well i.e. 1.00 × 10⁶ cells/mL. Then, 4 μL of 50 μg/mL anti-CD28 (previously diluted 10X in 10% CCM) antibody is added to coated CD3 wells. Plates are incubated at 37 °C with 5% CO₂ for 68 h, then alamar blue is added at 1:10 v/v and cells are incubated for 4 h (for a total of 72 h). The plate is excited at 560 nm in a fluorescent microplate reader and read at 590 nm. Data are expressed as fold increase (average OD₅₉₀ stimulated/average OD₅₉₀ unstimulated). Samples are assayed in triplicate, with intra-assay coefficient of variation < 10%.

Fatty acid composition of plasma and RBC

Total lipids in plasma and RBC phospholipids are extracted using the Folch method with a 4:1 ratio (chloroform: methanol (2:1, 8 mL):0.1 M potassium chloride (1.8 mL) [49, 53]. Samples are vortexed, incubated overnight at 4 °C and centrifuged (906 X g, 5 min with maximum brake) and the total lipids (bottom solvent layer) dried down under nitrogen gas and resuspended in boron trifluoride:hexane (1:1, 1.5 mL each) to methylate at 110 °C for 1 h. After cooling, 1 mL of deionized water is added, the samples incubated overnight at 4 °C and centrifuged as above. The total lipids (top layer) are dried down, then resuspended in 100 μL hexane for fatty acid methyl esters proportional analysis, using an Agilent 8,890 gas chromatograph coupled with an autosampler (7693A) using a 100 m × 0.25 mm × 0.2um CP-Sil 88 fused capillary column for long chain fatty acids. Identification of fatty acids is based on external GLC 643, GLC 502 and GLC 37 standards. The following fatty acids proportion will be determined in the RBC membrane and plasma at baseline, 3 and 6 months: octanoic/caprylic (C8:0), decanoic/capric (C10:0), undecanoic/undecylic (C11:0), dodecanoic/lauric (C12:0), tridecanoic/tridecylic (C13:0), tetradecanoic/myristic (C14:0),tetradecenoic/myristoleic (C14:1 n-9), pentadecanoic/pentadecylic (C15:0), hexadecanoic/palmitic (C16:0), hexadecenoic/palmitoleic (C16:1 n-9), (9Z)-hexadec-9-enoic/palmitoleic (C16:1 n-7), heptadecanoic/margaric (C17:0), 10-heptadecenoic (C17:1), octadecanoic/stearic (C18:0), octadecenoic/elaidic (C18:1 T n-9), octadecenoic/oleic (C18:1 n-9), 11-octadecenoic acid/vaccenic (18:1 T n-7), octadecatrienoic/α-linolenic (C18:3 n-3),octadecadienoic/linoleic (C18:2 n-6), eicosanoic/arachidic (C20:0), 11,14-eicosadienoic (C20:2n-6), 11,14,17-eicosatrienoic/dihomo-γlinolenic acid (C20:3 n-6), eicosatetraenoic/arachidonic (C20:4 n-6), eicosapentaenoic (EPA) (C20:5 n-3), tetracosanoic/lignoceric (C24:0),tetracosenoic/nervonic (C24:1 n-9), docosapentaenoic (DPA) (C22:5 n-3), docosahexaenoic (DHA) (C22:6 n-3). The fatty acid proportion is calculated as the fatty acid peak area/total peak area of identified peaks × 100 for each sample.

Plans to promote participant retention and complete follow-up {18b}

The participants’ regular visits to the HNRU plus phone calls will allow the study personnel to provide support, ensuring maximum adherence and motivation to adhere to assigned diets. Recipe booklets will be provided for each group to facilitate dietary adherence with inclusion of primary ingredients. Supplementary food provided at baseline and monthly follow-ups for a participant’s assigned diet will also encourage adherence.

Data management {26}

The research coordinator will ensure that checklists outlining all data to be collected from each study participant, have been completed. Study data will be collected and managed using REDCap electronic data capture tools hosted at The University of Alberta [54, 55]. REDCap (Research Electronic Data Capture) is a secure, web-based software platform designed to support data capture for research studies, providing 1) an intuitive interface for validated data capture; 2) audit trails for tracking data manipulation and export procedures; 3) automated export procedures for seamless data downloads to common statistical packages; and 4) procedures for data integration and interoperability with external sources [54, 55]. Data will be entered by trained personnel and independently verified. Data falling outside of expected ranges will be flagged and checked for accuracy.

Confidentiality {27}

All study personnel receive training in ethics in the conduct of research involving humans. Each participant will be identified by a unique alphanumeric code on documents and REDCap files. A master list of participants with their codes will be maintained by the research coordinator and stored on an encrypted, password-protected computer. The ASA-24 is accessed by a web link provided, with each participant receiving a unique username and passcode from the research coordinator; thus, their name is not linked to the ASA-24 database. Participants are provided a special study email address for routine correspondence. Any sensitive information is encouraged to be communicated by telephone. Only study personnel granted access by Alberta Health Services have access to the electronic medical records of participants. Access to identifiable information is limited to the research coordinator, study physicians and nutrition counsellors.

Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}

All biological samples will be stored according to standard operating procedures. Laboratory evaluations not conducted at the Alberta Precision Laboratories will be undertaken by trained personnel in a research laboratory at the ADI and associated core facilities (e.g. fluorescence-activated cell sorting), which are equipped for the relevant analyses. Any future use will be reviewed by the Research Ethics Board, which has already approved collection of feces for a planned future study.

Statistical methods

Statistical methods for primary and secondary outcomes {20a}

All results of the trial will be reported according to CONSORT guidelines. The main comparisons in this study will be intervention, time and the interaction between intervention and time. An intention-to-treat analysis will utilize data from all randomized participants who complete the two-week and one-month nutrition consultations; this will be considered as the primary analysis. A per protocol analysis will also be performed on participants who completed the two follow-up study visits at three and six months (study completers).

Descriptive statistics will be reported for all outcome measures at baseline, three and six months. Data will be tested for normal distribution using the Shapiro Wilkes test. Normally distributed continuous variables will be reported using means and standard deviations; those non-normally distributed will be reported using medians and interquartile ranges. Categorical variables will be reported using frequencies and percentages. Inter-group differences and within-group changes from baseline will be reported as means (medians) and confidence intervals.

Data will be analyzed in IBM SPSS Statistics version 29 [56] using an ANCOVA approach as described by Vickers and Altman [57]. For the primary and secondary outcomes, a general linear model with intervention and time as main effects, repeated measures, and adjusting for baseline values will be employed. In the intention to treat analysis, missing data will be dealt with by carrying the last value forward. The Tukey means statement will be used for multiple comparisons among groups. Statistical analyses will be adjusted for potential confounders in cases where groups are not well matched after randomization.

Interim analyses {21b}

Not applicable. Participants are monitored throughout (see 11b) and safety concerns are not anticipated.

Methods for additional analyses (e.g. subgroup analyses) {20b}

Participants will not be excluded based on adherence, but subgroup analyses will be performed to compare those who adhere to most nutritional recommendations for each respective diet based on 24-h recall questionnaires and the consumption of the food provided.

Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}

Intention-to-treat analysis will be conducted using multiple imputation in the case of higher than expected loss to follow-up and missing data (e.g. if the three-month follow-up is missing). All randomized participants will be included in analyses, with either observed or imputed outcome data.

Plans to give access to the full protocol, participant level-data and statistical code {31c}

The data will be available upon reasonable request to the PI.

Oversight and monitoring

Composition of the coordinating centre and trial steering committee {3d}

The KETO-IM trial does not have a formal coordinating center but trial personnel (PI, co-PI, co-investigator, study physician, research coordinator and graduate student) meet biweekly. Meetings with the study statistician are held on an as-needed basis. The research coordinator meets weekly with personnel performing data entry and verification.

There will not be a data monitoring committee for this trial considering the short timeline (duration of 6 months for participants, with enrollment across three years). We also expect the risks of this nutritional trial to be minimal. A recent study published by Roberts et al. on illnesses associated with ketosis during KETO diets reported that across 50,404 patient-years of follow-up in patients living with T2D, the incidence rate of diabetic ketoacidosis was 1.01 per 1000 person years [58]. Lab work will be regularly monitored for our participants by the study physician and acted on (through adjustments of medication or frequency of blood work) if required. There is no plan for an interim analysis for safety purposes.

Composition of the data monitoring committee, its role and reporting structure {21a}

Not applicable. This trial does not anticipate harm that necessitates prescribed data monitoring.

Adverse event reporting and harms {22}

There is a possibility that laboratory values or blood pressure assessments reveal unexpected findings, or that participants will report adverse events such as hypoglycemia. If the unexpected findings are associated with an increased health risk for the participant, they will be referred to the appropriate medical professional(s) by the study physician.

As no harms have been specified as primary or secondary outcomes, harms will be assessed by routine monitoring when participants are asked about changes to their health at follow-up at three and six months. Concerns will also be addressed at monthly follow-ups during nutrition counselling sessions, with the encounter charted on paper and kept with the participant’s file, or noted on REDCap. At the beginning of the study, participants are also advised to contact the PI or the study coordinator through provided phone numbers if participants believe they are experiencing any adverse effects or have suffered a research-related injury. Potential harms reviewed with participants prior to enrollment include the following: harms associated with blood draws (mild pain, syncope, bleeding, bruising, infection), harms associated with the KETO diet (hypotension, hypoglycemia, electrolyte disturbances, mineral deficiencies, constipation), ketoacidosis in the context of sodium glucose-linked transporter-2 inhibitors, and the need dose adjustments for insulin or other glucoregulatory drugs to mitigate the risk of hypoglycemia. The BIA measurement is a risk for participants only if they have a pacemaker or other internal electrical medical device due to the risk of device malfunction from a weak electrical signal.

To mitigate risks associated with blood draws, hand hygiene, gloves, 70% isopropyl alcohol, and sterile needles and syringes will be used. If infection occurs, the participant will be referred to one of the study physicians. Participants will be asked about allergies to latex, chlorhexidine, and isopropyl alcohol beforehand. Postural vitals will be monitored as needed, adjustments made to the participant’s body position, and hydration given for vasovagal syncope. Use of safety devices such as needle covers and tube holders that release needles with one hand, and safe practices involving sharps disposal will be followed to reduce sharps injuries (e.g. sharps container within arm’s reach, disposal of used sharps immediately). To reduce risks associated with following KETO, participants will undergo pre-diet evaluation and counselling, and have historical lab values reviewed to take corrective action if necessary prior to starting the diet. A multivitamin supplement will be provided to all participants, regardless of diet group, to prevent any potential nutritional deficiencies. Participants with T2D will be instructed to monitor their blood glucose. If patients are taking insulin, sulfonylureas and/or meglitinides when starting KETO, it will be recommended that dose reductions be made immediately to prevent hypoglycemia. The study physician will review and recommend ongoing medication and monitoring adjustments as necessary, with changes documented in REDCap. Participants will undergo frequent detailed medication review and medical supervision by an endocrinologist (i.e. the study physician). Participants with a pacemaker or other internal electrical medical device will be excluded from having their body composition assessed during the study. As optional stool samples taken from patients may contain infectious pathogens, participants will be given instructions on hand hygiene and safe transport and storage of stool samples.

There are no plans for formal analysis of harms data as they are not a primary or secondary outcome of the study considering the relatively safe profile of the study interventions. As mentioned previously, participants will be given instructions on how to transition away from their diet (if applicable), with provided resources for dietitian follow-up in the community if needed. If participants believe themselves to have suffered an adverse effect after the trial has ended, they will be directed to seek medical attention from the appropriate community health resources available to them (family doctor, emergency department, etc.), as adverse events that happen beyond the trial are outside the scope of the study for medical follow-up. However, participants will have a note in their medical records that future healthcare providers will be able to view indicating that they have been involved in the study.

Only adverse events that are serious and unanticipated will be reported to the University of Alberta Research Ethics Board. We will follow their standard protocol for reporting.

Frequency and plans for auditing trial conduct {23}

Not applicable. Considering the short duration of the trial and operation through a single site, there will be no formal auditing procedures.

Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}

Any protocol modifications must be approved by the Research Ethics Board at the University of Alberta. In the event that the modifications alter the eligibility criteria of individuals already enrolled, they would be informed by a telephone call from the research coordinator. Modifications would be communicated to the study personnel at the biweekly meeting and via email, and to ClinicalTrials.gov.

Dissemination plans {31a}

The funders place no restrictions on publication. In addition to academic journals, dissemination plans include relevant conferences in nutrition and diabetes, continuing education opportunities, and use of University media vehicles (newsletters, press releases) targeting the general public. The research team will work with Alberta Canola Producers and CanolaInfo.org as part of an integrated Knowledge Translation strategy. For example, inflammation assessed by circulating cytokines has been targeted as a unique outcome of KETO diet consumption that is not explored in most KETO trials. The Alberta Canola Producers has expressed particular interest in this outcome because of the uncertain effects of KETO diets on inflammatory outcomes given that they are generally high in SFA [18, 59].

Research participants are provided with their own study results at the end of their six-month intervention. At the conclusion of the trial we will provide a lay language summary to all participants via letter mail, or in-person pick up.

The research team will provide annual progress reports to the funding organizations. It will develop presentations as well as written reports aimed at the general public, physicians and other healthcare professionals to accompany the published manuscript(s) and conduct continuing education through the University of Alberta Faculty of Medicine and Dentistry. The conduct of this RCT will enable creators of nutrition guidelines such as Diabetes Canada and the American Diabetes Association to incorporate the evidence from the trial into new versions of guidelines. Indeed, Diabetes Canada published a position statement on the use of LCD for diabetes in 2020 [60]. The statement notes a concern that the quality of evidence was currently low. This research will help clinicians recommend evidence-based diets for individuals with overweight/obesity and with high glucose levels (prediabetes), and/or T2D. Through their professional contacts, guideline development and conference presentations, the research team will provide evidence that could influence the practice of their colleagues.

In addition, we will be training and mentoring personnel, including RDs and graduate students, who will enhance our ability to disseminate our findings through presentations, professional contacts and thesis work. We will also have the opportunity to mentor undergraduate students from the Dietetic Specialization and Nutrition Honors programs at the University of Alberta; these individuals will assist with data entry and analysis as part of their course requirements.

Discussion

The KETO-IM trial is a three-arm RCT designed to determine the effects of replacing SFA in a KETO diet with oil rich in MUFA, with the primary aim of mitigating cardiovascular risk. The study will compare three different diets for outcomes linked to cardiovascular risk and management of T2D. It has been shown that chronic diseases like T2D and obesity are often associated with low-grade inflammation and immune dysfunction. Thus, a novel aim of this trial is to compare the effects of each diet intervention on immune outcomes and determine the relationship of these effects to the systemic inflammatory profile. We also include measures to assess adherence related to actual consumption (dietary recalls) and metabolic outcomes (ketosis, TG).

Practice Based Evidence in Nutrition (PEN) recommendations state currently that there is no evidence supporting the effectiveness of the KETO diet over other diets higher in carbohydrate for long-term weight maintenance. PEN also states that currently there is not enough evidence to support any recommendations for use of KETO with individuals living with T2D based on low quality and heterogeneous information currently available [61]. Diabetes Canada, in their position statement [60], note that KETO may be one dietary option among many for some people with T2D, but also stress that there are knowledge gaps in both the safety and long-term sustainability of KETO. KETO is low in several micronutrients, including vitamin C, Vitamin D, calcium, magnesium and zinc, due to the restriction of whole grains, fruits and starchy vegetables [61]. There is a risk of nutritional deficiencies, if not adequately addressed. The KETO-IM trial will support all participants with nutrition counselling throughout the study period, with monitoring of diet recalls, and will provide a daily multi-vitamin to address potential micronutrient deficiencies.

Canola oil carries a health benefit statement, approved by the FDA, for its reduction of cardiovascular risk when included as part of a healthy diet. Part of this benefit comes from anti-inflammatory effects of UFA, like omega-3 fatty acids, found in canola oil [19]. It is recognized that individuals living with obesity and T2D generally have consistent levels of low-grade inflammation that can negatively impact an immune response and decrease individuals’ response to infection and illness [62]. SFAs are a ligand for the toll-like receptor-4, which produces an inflammatory response, worsening inflammation [63]. Alternatively, some studies report that the state of ketosis can dampen inflammatory responses due, in part, to the presence of circulating beta-hydroxybutyrate [64]. There is a knowledge gap, however, in our understanding of how the type of fat consumed in KETO impacts the inflammatory response and, in turn, how that influences immune function, including the response of T helper cells, which have important immunomodulatory functions [64]. We aim to understand if a KETO diet high in MUFA and PUFA, namely canola oil, will affect these responses compared to a KETO diet high in SFA and LFD, which is high in fibre. Interestingly, although the high-fibre MedDiet has generally exhibited anti-inflammatory effects, its ability to mitigate immune dysfunction is unknown.

The main limitation of this study is the short timeframe of six months. Several recommendations suggest that long-term data would be beneficial, however time and financial constraints often restrict a longer timeframe. As with most nutrition trials, neither the participants nor some study staff can be blinded to the intervention; however, the allocation and statistics are done by individuals not involved with participants. Trial registration and publication of the protocol, with a priori primary and secondary outcomes are measures to limit bias. Given the concern with increased LDL-C observed in other research with increased SFA intake [4], this trial will provide data comparing the effect of UFA intake while adhering to a KETO diet. Comparing the three diet groups in people living with, or at risk of developing, T2D, will provide data to inform nutrition recommendations in this population.

Trial status

The current trial protocol approved by the Research Ethics Board is Pro00123687, Version 4 (September 20, 2024). Recruitment began on July 1, 2023 and it is anticipated that recruitment will be completed by June 2026.

Abbreviations

ADI

Alberta Diabetes Institute

ApoB100

Apolipoprotein B100

BSA

Bovine serum albumin

CBC

Complete blood count

CCM

Complete culture medium

CRP

C-reactive protein

CVD

Cardiovascular disease

DMSO

Dimethyl sulfoxide

FACS

Fluorescence-activated cell sorting

FCS

Fetal calf serum

FMO

Fluorescence minus one

HbA1c

Glycated hemoglobin

HDL-C

High-density lipoprotein cholesterol

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HNRU

Human Nutrition Research Unit

IFN-y

Interferon gamma

IL

Interleukin

KETO

Ketogenic

KETO-Sat

Ketogenic diet enriched in saturated fatty acid sources

KETO-Can

Ketogenic diet enriched in canola oil

LCD

Low-carbohydrate diet

LDL-C

Low-density lipoprotein cholesterol

LFD

Low fat diet

LPS

Lipopolysaccharide

MUFA

Monounsaturated fatty acid

NK

Natural killer

PBMC

Peripheral blood mononuclear cells

PBS

Phosphate-buffered saline

PEN

Practice-Based Evidence in Nutrition

PHA

Phytohaemagglutinin

PI

Principal investigator

PUFA

Polyunsaturated fatty acid

PWM

Pokeweed mitogen

RBC

Red blood cell

RCT

Randomized controlled trial

RD

Registered dietitian

SFA

Saturated fatty acid

T2D

Type 2 diabetes

TG

Triglyceride

TNF-α

Tumour necrosis factor alpha

T reg

Regulatory T cell

UFA

Unsaturated fatty acids

Author’ contributions {31b}

JH: Writing-original draft preparation; Investigation. TG: Writing- Original draft preparation. PBC: Investigation, Project administration. CR: Conceptualization, Methodology, Writing – review and editing; Supervision, Funding acquisition. YM: Writing-original draft preparation; Investigation. AM: Writing-original draft preparation, Investigation. CBC: Conceptualization, Methodology, Writing – review and editing; Supervision, Funding acquisition. All authors read and approved the final manuscript.

Funding {4}

Funding for this study has been provided by Alberta Canola and Results Driven Agriculture Research (grant #2022F156R). CR receives salary support from the Canada Research Chair program (CRC TIER2 2023–00056). JH received a stipend award from Canadian Institutes for Health Research, Natural Sciences and Engineering Research Council of Canada, Social Sciences and Humanities Research Council and the Alberta Diabetes Institute. TG received a stipend from the Lorna Bradley Diabetes Research Endowment, and the University of Alberta. The funders have no involvement in the study design or the collection, analysis, and interpretation of data. Decisions regarding report writing and publication of data are made solely and independently by the study team. The primary investigators and research coordinator are salaried employees of the University of Alberta. Experiments performed at the University of Alberta Faculty of Medicine and Dentistry Flow Cytometry Facility (RRID:SCR_019195) receive financial support from the Faculty of Medicine and Dentistry and Canada Foundation for Innovation.

Data availability {29}

Data will be made available upon reasonable request to the Principal Investigator.

Declarations

Ethics approval and consent to participate {24}

University of Alberta Research Ethics Board Pro00123687. Written, informed consent to participate will be obtained from all participants.

Consent for publication {32}

A model consent form is available upon request.

Competing interests {28}

The authors declare that they have no competing interests.