Study design and setting
This is a double-blind, parallel randomized controlled trial for central obesity participants. The Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) 2013 statement will be followed in this trial (Additional file 1) . The proposed clinical trial will be conducted at the Community Health Service Centre (CHNS) in Nanshan District, Shenzhen, Guangzhou Province, China, for 12 weeks to assess the effects of lutein supplementation (10 mg/d) on inflammatory biomarkers and metabolic risk factors in adults with central obesity. Figure 1 illustrates the overview of the study.
Study participants and eligibility criteria
It was estimated that 52 participants per treatment group would be required for 80% power and 5% level of significance to detect the change of serum TG over 12 weeks of supplementation . Allowing for a dropout rate of approximately 15%, we aim to recruit 120 participants in total. Participants were eligible for this study if they fulfil the following criteria (1) aged between 18 and 60 years; (2) waist circumstance: male ≥ 90 cm and female ≥ 85cm; (3)deficient in lutein intake (dietary lutein intake＜10 mg/d) or low levels of serum lutein; (4) willing to sign the informed consent. People with any of the following will be excluded from participation: (1) using lutein supplementation or related health care products with 1 month; (2) using antibiotic in 1 month; (3) secondary obese patients; (4) those who had a special diet or meal replacement for early 2 weeks; (5) using insulin or insulin sensitizers in 1 month; (6) people with chronic diseases (including type 2 diabetes, hypertension and dyslipidemia) over 10 years; (7) patients with hepatorenal dysfunction (creatinine > 1.2 times the upper normal limit of creatinine, aspartate aminotransferase > 1 times the upper normal limit); (8) any active tumors/cancers; (9) Regular use of drugs affecting blood lipid; (10) Pregnant or lactating women; (11) history of gastrointestinal surgical operation;
Randomization and blinding
When all the inclusion and exclusion criteria have been executed and informed consent have been obtained, participants will be allocated to lutein group or the placebo group. Computer-generated random numbers will be used for allocation sequence generation with stratification completed according to gender, age and waist circumstance. This parts of work will be perform by a persons who will be operationally independent from the study team. Allocation concealment will be implemented by using the random number card in a black envelope. The investigator and patients will be blinded to the sequence and the treatment condition. Patients’ data collected will be kept confidential during this trial. The grouping of the study will be opened after all individuals complete the study protocol.
The participants will be receive either 10 mg of lutein or placebo capsules per day for 12 weeks. Lutein were suspected in soybean oil and given in acid soluble gelatin capsules and the placebo containing soybean oil (100%). To maintain and guarantee blinding, the capsules will be identical in appearance, shape, smell and weight. All capsules will be provided by the Royal DSM N.V. (Beijing, China). Face to face instruction on how to take the capsules will be provided at the baseline. Enrolled participants will be required to: (1) take one capsule along with meals daily; (2) bring the remaining capsules in the bottle to the next visit; (3) avoid to make any changes in dietary habits, such as suddenly eating plenty of foods rich in lutein. (4) avoid taking any health care products during follow-up.
Recruitment and screening
Table 1 provides the schedule for enrollment, intervention and follow-up. Participants will be recruited by placing advertisements on social media sites and community notice boards. Once the volunteer expresses initial interest in participating in the study, he/she will have a face to face or telephone communication with the research assistant. Detailed information on the study trial including the purpose, the way of interventions and durations will be told to the volunteers. If they are still interest in this project after careful consideration, the structured standardized screening questionnaire, which is mainly around the inclusion and exclusion criteria for the study, will be used to assess the potential eligibility of the participants for the study. Potential participants who are found not to be eligible will be excluded at this stage. Eligible participants will be invited to attend the study for a further screening visit, which takes place at CHSC. Firstly, the informed consent will be obtained from all participants. Second, for safety assessments, the liver and renal function will be evaluated by collecting fasting blood samples and spot urine samples. Third, the anthropometric index will be measured by the trained investigators.
In the test visit 1 (week 0), eligible participants are allocated to either a lutein supplement group or a control group. The lutein supplement group involves one 10 mg lutein capsule daily and the control group takes the placebo. They will be given the 6 weeks supply of capsules in the test visit 1 and visit 2, respectively, and required to return the remaining capsules in the next visit, which will be counted and recorded for each participant in order to ensure compliance with the protocol. Participants are defined as non-compliant if they have been taken less than 80% of the study product. Medication, health status or any recent changes around the inclusion and exclusion criteria since screening questionnaire are also re-assessed in order to evaluate whether participants are still suitable to participants in the study. Anthropometric measurements and blood sample will be collected at each visit, and stool sample, spot urine sample and dietary assessments will be collected at test visit 1and test visit 3. At the test visit 1 and test visit 3, the volunteers are asked to complete the short form international Physical Activity Questionnaires (IPAQ-short form) and Mini-mental State (MMSE). Throughout the intervention, each patient will be advised not to make any changes in her/his dietary habits and physical activity level (PAL). At the end of the each visit, participants are thanked for their time and will be provided the breakfast. After the test visit 2, people in the placebo will be given the same amount of lutein supplements as the intervention group. Full details of data collected at each time point can be found in Table 1.
Anthropometric measurements will be collected using standardized equipment and examination procedures. Participants will be weighted and heighted with light clothes and without shoes in the morning of the follow-up. Body fat percentage and total body water will be calculated using one-stop self-service monitoring machine (E-Techco Information Technologies Co., Shenzhen, China). Waist circumference (WC) is measured at the midpoint between the lower edge of the rib arch and the iliac crest while the subjects are standing with steady breathing. The hip circumference (HC) will be measured at the widest part of the buttocks at the intertrochanteric levels to the nearest 0.1cm. The other anthropometric indices will be calculated using the following equations: BMI=weight (Kg)/height (m2); waist-to-hip ratio (WHR) = waist (m)/hip (m); waist-to-height ratio (WHtR) =waist (m)/height (m). Blood pressure (BP) will be measured using a standard mercury sphygmomanometer with a cuff placed on the upper right arm after a 5-min rest with a 1-min interval before subsequent measurements. Three BP readings are recorded, and the mean will be calculated. All anthropometric measures will be taken by trained research assistant using standard equipment according to the standard guidelines.
At the baseline, middle and the end of the trial, 12 ml of venous blood sample (4 ml in EDTA-coated sterile tubes and 8 ml in regular tubes) will be collected after fasting overnight. In addition to the indexes of liver and renal function (alanine aminotransferase (ALT) and aspartate aminotransferase (AST), creatinine), blood glucose, insulin and lipid profile (TC, TG, high-density lipoprotein cholesterol (HDL-C) and LDL-C) and CRP will be test on the day of the blood collection, the remaining samples will be divided and stored at -80°C freezers for long-term storage. The enzymatic methods (Biosino Biotechnology Company Ltd., Beijing, China) will be used for measuring serum hepatic enzymes, including the alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Serum creatinine will be determined using the Jaffe methods. Insulin concentrations (μU/mL) will be measured with Chemiluminescence microparticle immunoassay, high-sensitive CRP will be measured using the immunoturbidimety assay, fasting blood glucose (FBG) (mmol/L) will measured by using the enzymatic colorimetric method with glucose oxidase using commercially. Insulin resistance (IR) will be estimated by homeostasis model assessment of insulin resistance (HOMA-IR): fasting insulin (μU/mL)*(fasting glucose (mmol/L)/22.5). Lipid profile will be determined by colorimetric methods using commercial kits (Biosino Biotechnology Company Ltd., Beijing, China) in Hitachi 7080 automated analyzer (Hitachi, Ltd., Tokyo, Japan). Serum lutein (µmol/L) will be analysed by high-performance liquid chromatography-tandem mass spectrometry method, retinol-binding protein 4 (RBP4) (μg/mL) and adiponectin (μg/mL) will be measured using the enzyme-linked immunosorbent assay (ELISA) kit (Adipogen, San Diego, CA, USA). Serum levels of MCP-1, TNF-α, IL-6, Interleukin-1β (IL-1β) will be measured using the flow cytometry method (FCM).
Participants will be required to provide the spot urine samples on the day of the screening, test visit1 and test visit 3. The routine urine test will be executed on the day of collection and the remaining samples will be randomly packed in 5-ml EP tube and then stored for future analysis. Fecal samples will be collected at the test visit 1 and visit 3 using a stool specimen collection kit, which includes the operating instruction, an ice pack, gloves, one sterile container and spoon, sealed plastic pouch and a cool box. Participants can collect their fecal samples and bring them to the CHSC and then the samples will be transport and stored for further analysis.
At the screening periods, eligible participants will complete a questionnaire on food intake, which capture information on how much vegetables rich in lutein they intake, including the sweet potato leaves, spinach, broccoli, collards, leeks, lettuce and so on. This questionnaire provide a reference to roughly evaluate dietary lutein intake. At the baseline (week 0), middle (week 6) and the end of the trial (week 12), individuals will be required to provide the three days of 24-h recall through the phone or the internet. The data will collected by and calculated by the professional nutritionist according to the Chinese Food Composition Database (2009) and the United States Department of Agriculture Nutrients Database [27-28]. At each follow-up, questions on whether they are have changed their diets will be asked, including significant reduction or increase in dietary intake.
The primary outcome is to measure changes to the inflammatory biomarkers between the intervention and placebo group from baseline (test visit 1) to 12 weeks later (test visit 2). The secondary objectives are to assess the change of the anthropometric index, including the weight, waist circumference, hip circumference and the body composition.
Side effects and safety assessment
There are no side effects of 10 mg/d of lutein supplementation. The observed safe level (OSL) of 20 mg/d for lutein has been suggested . All participants can contact research assistant at any time if they feel any uncomfortable. In addition, we will always pay attention to gastrointestinal symptoms and other adverse effects by phone calls or the internet. In case they develop severe side effects, they are advised to stop to take the supplementation. All serious and non-serious adverse events and/or reactions involving capsules supplementation are record by the research assistant. Individuals are reminded of the changes they have been asked not to make and they have been requested to record timely once these behavior had been changed.
Two population will be used in the analyses. The intention-to–treat (ITT) population includes all participants who have been randomized, while the per-protocol (PP) population contain all participants who accomplish the entire intervention. Baseline characteristics of the study will be summarized as means± standard deviations (SDs) for parametrically distributed data, geometric mean values (and 95 percent confidence intervals) for nonparametrically distributed data, and numbers (percentages) for categorical data, respectively.
Difference between participants who complete and withdraw from the trial will be analyzed using an independent t test or the Mann-Whitney test for continuous variables (e.g. age) and chi-square for categorical variables (e.g. gender). For primary and secondary outcomes, analysis of covariance will be used to examine differences between lutein supplement and placebo at 12 weeks, adjusting for potential confounding factors and effort modifiers (e.g. baseline age and gender, etc). Our statistical analysis will be performed by using R software and the results will be considered significant at P＜0.05.
Individuals’ data or other related information will be kept by in a located file at the principal investigator office. The authorized study team will enter the information into electronic database using the double-entry methods. The private personal information (e.g. ID number and telephone) will be hidden and restricted to principal investigator. The electronic database will be unified managed by Shenzhen Nanshan Center for Chronic Diseases Control.