Study Protocol

Study Title- Impact of “GUUD test Product “on Glycemic variability and Metabolic Health compared to normal sugar (Sucrose) in obese diabetic and non diabetic patients; A randomized open label two arm two phase crossover design Study – GUUD Health Study.

Running Title- GUUD Health Study

 Introduction

India is facing a potential healthcare crisis, owing to rising obesity associated with poor metabolic functions and glycemic variability, significantly shifting to type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD), hypertension, cancer, kidney disease, neurological disorders and nonalcoholic fatty liver disease. Obesity and T2DM (caused by high calorie intake, particularly in the form of sugar rich diet and sedentary life style) are the major contributing factors in further accelerating metabolic illness and non communicable diseases (NCDs). The consumption of sugar and sweets has been prominent in Indian culture, rituals, and religion. In the year 2000, the amount of white sugar consumed per person/day was 22 g/day, which had increased to approximately 55.3 g/day in 2010, if we match same propensity ratio, present sugar intake may be around 80-100g/day nationwide. Moreover if intake from traditional sugars and sugar-sweetened beverages (SSBs) is added, it is more than the worldwide per capita consumption of 23.7 kg per year. Sucralose, aspartame, saccharin are some of the commercially available artificial sweeteners which are widely used as calorie-free alternatives to sugars. Recent studies suggested that, in long term artificial sweeteners contribute to weight gain and hence increase the risk of metabolic syndromes and NCDs.

World Health Organization (WHO) recommended, free sugar intake should be decreased to less than 10% of total calorie intake. Lowering free sugar might be a revolutionary way to improve metabolic functions by reducing energy consumption. Dietary modifications with low-calorie sweeteners (LCS) as sugar substitutes can help in improving cardio metabolic risk. Furthermore, such calorie reductions, accomplished without altering taste are anticipated to result in increased dietary compliance and better weight management and long term adherence. Natural LCS, steviol glycosides, have gained immense popularity and long term acceptability. Besides, safety, stability, wide commercial availability, it provides no calories and also has antioxidant properties.

Steviol glycosides, derived from the leaves of Stevia rebaudiana Bertoni, are natural, sweet-tasting, calorie-free natural sweetener that may be used as a sugar substitute. Compared with sucrose, stevia is approximately 200 times sweeter. High-purity steviol glycosides are generally recognized as safe and are approved by the US FDA and the European Food Safety Authority for human use. Clinical evidence suggests that steviol glycosides have positive effects on insulin sensitivity and postprandial glucose levels in humans with T2DM. However, limited evidence is available on the effect of steviol glycosides on metabolic health, glycemic variability in overweight/obese diabetic and nondiabetic subjects, especially in the Indian population.

Study Background

Recent WHO sugars guideline recommends that adults reduce their intake of added sugars to <10% of total energy intake and a further reduction to <5% for additional health benefits, to halt the increase in diabetes, obesity, and premature deaths by 25% by 2025. The UK Scientific Advisory Commission on Nutrition also recommends a reduction in free sugar to ≤5%. In India present high consumption of sugar with increasing trend, added in a variety of foods and beverages, predisposing them to metabolic ill and further contributing for NCDs. Substituting high sugar intake (>10% of total calorie) with low calorie product matching the same magnitude of sweetness as table sugar can be an important primary preventive strategy to combat high burden of obesity and metabolic syndrome in India.

In present study, the test product which shall be used for intervention is “GUUD” which is a novel product manufactured with sugar and stevia combination, it is 50% lower in calories when used as directed. GUUD as sugar crystal provides the same sweetness with half the amount of table sugar. In GUUD, high-purity stevia is extracted and purified from stevia leaves in a manner that is similar to that of sucrose from sugar cane. Stevia contributes to zero calories, offers better compliance. Moreover, it is an effective alternative for people who are habituated to high sugar intake with an insight into a healthy lifestyle. Presently only few clinical studies were conducted to evaluate the efficacy and safety of stevia-based table top sweetener formulation when used to replace added sugar in the diet among obese patients. Current study is designed to evaluate the efficacy of “GUUD test product” for improving metabolic health by completely replacing table sugar in adult obese patients with and without diabetes

Study Rationale and Significance

In India, any celebration is traditionally marked by the consumption of sweets. After every meal, any happy event, religious holiday, social gathering, etc., it is a tradition to “sweeten the tongue”. People in India are at an elevated risk of metabolic disorders owing to a higher metabolic load. It is believed that for every given level of BMI, there is a proportionally higher ratio of fat to lean mass, and as BMI rises, the metabolic load rises too. Therefore, preventing excess weight gain, abdominal obesity, and sedentary behavior, as well as a transition to low-glycemic index is necessary. A diet that can self-regulate dietary energy intake can play a key role in long-term improving metabolic functions. Excess sugar consumption leads to the buildup of body fat and intra-abdominal fat, increased insulin resistance, excess liver fat, hypertriglyceridemia, increased free fatty acids, contributes to the development of T2DM, CVD and associated mortality.

Non-nutritive sweeteners can help people cut back on added sugars, resulting in calorie reduction and weight loss, as well as positive impacts on metabolic functions. The American Heart Association (AHA) and the American Diabetes Association (ADA) issued a joint statement saying that stevia and comparable sweeteners can help to improve metabolic functions in obese patients, provided they use them correctly and do not compensate by consuming more calories later in the day. Sugar restriction while retaining natural taste and appearance in products can be a revolutionary concept for long term adherence. GUUD test product has a balanced formulation of natural sweetener stevia and sugar in a proportion to produce the same sweetness of sugar at 50% consumption, as instructed. The present study design to confirm the metabolic benefits of GUUD test product in obese patients with and without diabetes in rationale proportion.

Study design and methodological

The effects of substituting GUUD test product matched with same magnitude of sweetness for table sugar among adult obese patients with and without diabetes shall be assessed at Jaipur center in Rajasthan, India. To assess the effectiveness of GUUD test product as substitute of normal sugar, a randomized open label two arm two phase crossover design method shall be adopted.  Total 56 obese (BMI≥25 Kg/M2) patients with and without diabetes shall be recruited in the study in equal proportion (26 diabetic and 26 nondiabetic). In first phase, participants shall be   randomized into two intervention arms in equal distribution (26 diabetic and 26 nondiabetic) for three months. At baseline after assessing total monthly consumption of sugar, quantity matched GUUD test product shall be allocated as replacement of sugar for one month period. All subjects in both diabetic and nondiabetic groups shall visit at first month, second month for follow up, so that assessment of monthly consumption as well as further reallocation of GUUD test sample for next month can be done. In second phase, completing a two-week washout period after third month follow up, responders  in both diabetic and nondiabetic will switch to normal sugar consumption  in a single arm crossover design mode for further next 3 months. Subjects in both groups shall be allow to use normal sugar in a similar frequency and quantity as they were using prior to enrollment in the study. Patients in both groups have to present at investigation site at for monthly vist at fourth and fifth month. Final follow up will be performed at sixth months in both groups.  In study subjects all prescribed physiological, clinical and biochemical parameters shall be recorded at baseline, third month in first phase, in second phase at baseline (after completing 15 days washout period and 3rd month follow up in first phase) and at six month in both groups.

Continuous glucose monitoring (CGM) device will be used to assess glycemic variability for alternate subjects for two weeks in both groups, under the guidance of clinical investigator or treating physician.

            All the recruited participants will get details about the purpose of the study, benefits, risks involved and the importance of the participants’ full cooperation. These details are also included in the consent document, which each participant will read and sign before entering into the study. A one-week run-in phase (in pilot mode) is planned, that will help to assess the conduction of the study in line of the defined protocol prior to the start of the intervention. It will also help to evaluate participants’ compliance to the study protocol and to monitor any potential noncompliance factors and adverse outcomes. The study protocol will obtain approval by the Institutional Review Boards of EHCC hospital, Jaipur, before recruiting patients.

To avoid any bias between groups and to get maximum effectiveness of intervention, screening, randomization and dietary counseling will be performed separately. At baseline all subjects in both shall be advised to perform mild to moderate standard physical activity approximately 40 minutes at least 5 times in a week and same need to record in prescribed diary, so that the   impact of physical activity can be assessed in outcomes. Patients in diabetic intervention group, recommendation of physical activity and use of GUUD sample will be performed in strict clinical supervision to avoid any hypo or hyperglycemic events. Two separated Whatsapp groups (diabetic and nondiabetic) shall be created to monitor progress, immediate response to queries, clinically adverse outcomes, sharing experience among patients, inter personal motivation, delivery of prescribed educational input at defined interval, follow up schedule intimation and to maintain appropriate regular communication with team members.  Present study design involves a real-life scenario, and participants shall be allowed to make choices and flexibility to adjust the addition of the GUUD test sample frequency and quantity in a proportion to their daily routine requirement. This makes this study realistic.

Primary Outcomes

To assess impact of GUUD test product on glycemic variability and metabolic functions, compared to table sugar among overweight/obese subjects with and without diabetes by using a randomized open label two arm two phases, cross over design method.

Primary outcomes

To assess effectiveness of “GUUD test product” compared to quantity match normal sugar in improving glycemic variability and metabolic function in overweight/obese diabetic and nondiabetic subjects by assessing biochemical investigations.

Secondary Outcomes

 Secondary outcomes shall be reported by analyzing changes in BMI, waist circumference, hip circumference and cardiovascular risk.

Study Population

Total 52 obese patients with and without diabetes (BMI≥ 25 kg/m2) between the ages of 18- 65 years shall be recruited in the study, equally distributed in two groups (diabetic & nondiabetic). Eligible participants, who will agree to participate in the study, will be randomized after a written consent into any one appropriately assigned category among two groups. (I) group: nondiabetic overweight/obese (n=26), group (II): diabetic over weight/obese (n=26) by using appropriate randomization method. In first phase design all patients shall be equally randomized in both diabetic and nondiabetic group who will adopt suggested life style modification and GUUD sample as a substitute of normal sugar. In second phase both diabetic and nondiabetic groups after 3 month follow up, subjects will be allowed again to take normal sugar with continuation of suggestive lifestyle. Between two phases at least a 15 days washout period shall be maintained to wipe out any residual effect and to get maximum effectiveness of the intervention applied.

Prescribed physiological and biochemical assessment shall be reported at baseline, third month follow up, second stage baseline and at 6 month follow up.

Phase-I: Randomization at baseline

Group (I):  Nondiabetic overweight/obese patients who will substitute ordinary sugar with GUUD sample product in their diet with life style modification.

Group (II):  Diabetic overweight/obese patients who will substitute ordinary sugar with GUUD sample product in their diet with life style modification

Phase-II: Responders after completing third month follow up will undergo a 15 days washout period.

Group (I):  Nondiabetic overweight/obese subjects will again resume ordinary sugar consumption in their diet with continuation of life style modification.

Group (II):  Diabetic overweight/obese patients will again resume ordinary sugar consumption in their diet with continuation of life style modification

Participant recruitment and eligibility criteria

Participant volunteers shall be selected based on the inclusion and exclusion criteria as shown below.  Patient’s selection shall be done both from OPD and directly from community. A testing session of intervention sample GUUD test product, awareness talk with products’ brochures emphasizing the possible health benefits of GUUD test product and an introduction to the intervention shall be done prior to screening.

Inclusion criteria:

1. Overweight/obese patients with or without diabetes (BMI≥ 25 kg/m2) between ages 18-65 years.

2. Participants who are willing to provide written informed consent.

3. Participant who are willing to follow suggested dietary intervention, and other prescribed guidelines.

Exclusion criteria:

1.      Patients with type 1 diabetes.

2.      Type2diabetic with severe complication like; heart failure(grade 3-4), CKD etc

3.       Patients with advanced renal, hepatic or cardiac disease, cancer.

4.      Patients with genetic predisposition to metabolic disorders.  

5.      Any history of hypersensitivity with stevia glycoside and other components in the test products or an adverse reaction that might affect the study.

6.       Women who are pregnant or lactating.

7.      Refusal to sign an informed consent.

Intervention plan and follow up schedule

Randomization shall be performed using computer generated random numbers adjusted for gender, age and BMI to avoid differences in the proportion between groups for achieving optimum equipoise level. To assess the exact quantity of sugar consumption by an individual, a 30 minute session on current food pattern and proportion of sugar used shall be evaluated and validated by study team members using standard dietary questionnaires. Exact daily use of sugar would be derived and extrapolated in similar proportion for allocation of GUUD sample quantity.

To monitor whether the participants are receiving the prescribe frequency and adequate quantity of GUUD test product or table sugar items; it shall be recorded in diet diary at regular interval. Preparations of both GUUD test product and table sugar items based recipes shall be quantitatively standardized at individual level. Prior to the beginning of the intervention, a 2-week run-in phase for training and validation of energy products to be used in both group would be done. Logistic support, noncompliance and nonadherence determinants would be assessed. Before starting intervention training session shall be conducted to evaluate participant’s motivation, and willingness to adhere to the diet throughout the study period.

In phase-1, patients in both the groups shall be asked to replace normal sugar with a standard amount of GUUD test product in food preparation and beverages such as tea, coffee, smoothies, milk etc and the amount consumed shall be properly noted in the applied intervention tool to verify it further. Amount of GUUD test product assigned in both groups shall be cross matched and validated with total monthly sugar consumption by participant at individual level. Delivery of GUUD test sample in both groups shall be at baseline, 30 days, 60 days and 90 days in first phase and same chronology will be followed in both groups with normal sugar consumption after completing washout period.  

During follow-ups, at 30 days, 60 days and 90 days, completed diet diary shall be checked to ensure compliance, and subjects will be asked to adhere to the prescribed guidelines. Subjects will be asked to follow a mild to moderate physical activity (40 minutes for at least 5 days a week) in both arms. At baseline, in both the groups demographic details, comorbid physical parameters, biochemical assessment condition shall be reported. Physiological parameters need to be assessing within intervention group at, 30 days and 60 days follow up. After 90 days all physiological and biochemical parameters shall be recorded in both groups. Additional sugar substitute of any other kind will not allow other than the GUUD test sample in first phase in both groups. Continuous glucose monitoring (CGM) device will be used to assess glycemic variability for alternate patients for two weeks in both groups, under monitoring of clinical investigator or treating physician.

Sample size justification and study power

In this study by using GUDD test product, reduction in sugar calories without compromising the sweetness, can help in improving metabolic function, glycemic variability and weight loss which are interdependent. Present study has been designed to detect a significant difference in glycemic variability and metabolic outcomes by using GUUD test sample in diabetic and nondiabetic overweight/obese patients compared to quantity matched normal sugar as the primary outcome. Studies have reported losing almost 1.7 times more weight in stevia containing group than ordinary sugar group.  Assessment of previous similar intervention studies composite outcomes reported at least 2.5 kg weight reduction and 3-8 % improvement in waist circumference to hip circumference ratio (WHR) from their baseline after 90 days when stevia based test product was used as part of a daily balanced diet. In the present study we assume minimum 5% weight loss and 3-5% improvement in WHR after intervention from baseline, extrapolating same propensity match physiological expected outcomes, it will provides at least 8-10% improvement in glycemic variability (0.5% reduction in HbA1c) and metabolic outcomes in both groups compared to normal sugar used at 3 month follow up. To get adequate power for similar outcomes, total 52 patients, 26 in each group shall be needed. That will provide 80% power to detect these changes, even if a 20% dropout occurs at 3 months & 30% dropout at 6 months final follow up. Further, two arm two phases crossover design and follow up at next 6 months, will minimize residual effect and study further empowered for high generalizibility to achieve primary outcomes. The effect of confounding factors such as physical activity will also be taken into consideration. It has power to assess relation for glycemic variability and metabolic outcomes with lower empty calorie intake in individuals with overweight and/or obesity. Present sample size, doesn’t have power to detect secondary outcomes like cardio-metabolic event.

Statistical analysis

In study effect of intervention to be evaluate by comparisons from baseline using linear mixed model for continuous variables and a binary or ordinal mixed model for categorical variables, in each case treating as the random effect. Continuous variables shall be reported using descriptive statistics (mean, standard deviation, median, and range), whereas categorical variables shall be reported by using count and percentage of subjects. All comparisons within and between groups shall be  performed using two-tailed paired t-tests (before and after the intervention) and independent sample t-tests and among groups by using ANOVA test with a significance threshold of 5%, respectively.  Mixed-model repeated measures ANOVAs shall be used to assess the glucose and insulin responses.. All statistical analyses shall be performed using SPSS, version22 (IBM, USA).

To maintain a systematic data recording of patients anthropometric, dietary, biochemical parameters and to estimate the dietary records of the participants at baseline, follow up period and at the end of the stud in house software shall be used for effective intervention, response rate and to manage proper monitoring of patients in study cohort. Participant confidentiality shall be assured through a computer-based data coding system that de-identifies the data before analysis. Adequacy of the randomization process shall be assessed by comparison of baseline characteristics. Differences in changes from baseline between intervention and standard care arms in biomarkers, body measurements, and dietary intake, shall be calculated using the optimally efficient statistical tool.

Patients monitoring and follow-up

Participants in the interventions group shall be contacted directly by study team members every month to keep them motivated and for continuation in the study. At baseline, 3 months and at 6 months, all physiological and biochemical parameters shall be assessed. Participants who discontinue the study shall be contacted and their reasons for discontinuation shall be recorded. All patients in both groups have to be visit hospital for monthly follow up visits at first month, second month in first phase and fourth and fifth month at second phase for effective monitoring of suggestive life style modification and current dietary pattern. To assess compliance, participants will record their daily dietary pattern, number of sweet meals consumed, and serving sizes of foods consumed in study diary. Participants will be informed about possible adverse effects due to consumption of GUUD such as diarrhea, flatulence, nausea, and/or bloating early in the trial period. If any serious side effects of the interventions are detected, participants shall be withdrawn from the study.

For feasibility reasons, the intervention shall be ad-libitum (prescribed dietary frequency and quantity) mode, trying to restrict to one breakfast followed by 2 meals a day per week, in between if energy beverage and snacks are added (with empty energy contents) it would be recorded in both groups.

Baseline screening questionnaire

A screening questionnaire shall be developed for all participants during recruitment to assess their usual sugar intake currently, medical history, demographics, and lifestyle factors such as physical activity, alcohol consumption, and smoking status. Upon completion of the questionnaire, a checklist of inclusion and exclusion criteria shall be used to qualify participants. A validated food frequency questionnaire, adopted from PURE (Prospective Urban and Rural Epidemiological, standardized in Indian population) study shall be applied to assess the dietary habits of participants prior to randomization to evaluate their baseline dietary pattern, monthly sugar consumption and weekly physical activity status. Each participant shall be advised to record 24-hour dietary components daily during the follow up period. The participants in second phase will not be required to change anything in their usual diet. Both groups shall be advised to keep their physical activity levels consistent for the duration of the study period.

After screening all eligible participants and completing food pattern questionnaire to validate individual based sugar consumption, those who fulfill the inclusion criteria shall be asked for physiological and biochemical assessment at baseline. Eligible participants agreeing to participate shall be consented and then randomized into one of the study groups.

Anthropometrics

Body weight (kg), height (cm) and waist circumference (cm) be measured according to standardized procedures. Body mass index (BMI) shall be calculated as the weight (kg) divided by height (meters squared). Obese are defined as those having a BMI≥25 kg/m2.

Laboratory

The baseline biochemical parameters shall be assessed, blood samples shall be obtained at baseline and at the end of each intervention (after 3 months and 6 months) and analyzed at a NABL accredited lab. The samples shall be immediately centrifuged, aliquoted, transported on appropriately and stored. Laboratory technicians shall be blinded to the participant’s study group. Hb, plasma glucose, HbA1c, lipid profile, s.creatinine s. insulin, ALT, hs-crp will be done at beginning, end of first 3 months and  end of the study at 6 months in all subjects. Continuous glucose monitoring (CGM) by US FDA approved CGM device (Abbott- Libre pro) will be done on every alternate subject along with the lab tests in the same frequency. We intend to see the change in glycemic parameters, insulin resistance, inflammatory marker and lipid profile. Glycemic variability will be assessed by CGM.

Safety Evaluation

The safety of the test product (GUUD) in intervention subjects shall be evaluated and reported. The clinicians (investigators, i.e., medical doctors) will further assess the safety by recording any adverse reactions reported by any subjects during the study period.

Study Flow Chart

Effect of Steviol glycosides reported on health and related biomarkers

1. Postprandial blood glucose and insulin effects

It is well established that the intake of sucrose or glucose creates a postprandial increase in blood glucose and insulin. Three randomized controlled trials observed a significant reduction in postprandial blood glucose with purified steviol glycosides utilized in reduced-sugar/calorie meals or in supplement form in healthy subjects and diabetics. Despite no sugar, carbohydrate, or calorie difference between the test groups, stevioside significantly reduced postprandial blood glucose. There was a trend toward an increased insulin response and a 40% increase in the insulinogenic index when stevioside was consumed compared with Placebo. Overall differential, postprandial blood glucose reductions have been observed, and this effect is largely due to a sugar and calorie substitution.

2. Fasting blood glucose and insulin effects

Long-term studies indicate that high-purity steviol glycosides in supplement form within interventions that have no dietary carbohydrate or calorie manipulation do not significantly reduce fasting blood glucose, insulin, or glycated hemoglobin (HbA1c) concentrations.

3. Blood pressure

A meta-analysis of randomized controlled trials that assessed steviol glycosides in both acute single-meal and long term settings showed a nonsignificant difference in systolic blood pressure but a significant decrease in diastolic blood pressure.

4. Gut microbiota

The human gut microbiota is a large and complex population of microorganisms. There is also evidence that the microbiota may also be involved in obesity and type 2 diabetes. Presently no strong evidence suggest impact of steviol glycosides on gut microbiota.