Study Design and Participants
In a single-blind, randomized, controlled study design, eligible participants who provide informed consent are randomly assigned into a LC/HP dietary intervention group or a control group in a 1:1 ratio. Randomization is performed using the block randomization method (block size 4) by the study statistician (RO). A randomization list is generated, and assignments are placed into closed envelopes and given to each study participant by the study coordinator (JL). The study statistician is blinded to the group assignments. A Standard Protocol Items:
Recommendations for Interventional Trials (SPIRIT) schedule is presented in Figure 3 and the SPIRIT checklist (additional file 1). Outcome measures are performed at two time points: at baseline, before starting the LC/HP dietary intervention; and after completion of the 8-week LC/HP dietary intervention (post-study assessments are performed the morning after the last day of the intervention). A summary of data collection schedule for main outcomes is shown in
The study population is expected to be representative of demographics of the spinal cord injury (SCI) patient population. Based on 2017 Spinal Cord Injury Facts and Figures, 81% of SCIs reported to the national database have occurred among males (2). Therefore, we anticipate that among those who participate in this study, 81% will be male and 19% will be female. SCI is more common in non-Hispanic whites (63% non-Hispanic white, 22% non-Hispanic black, 11% Hispanic origin, 1% Native American, 2% Asian, and 1% Other); therefore, we expect that more non-Hispanic whites, as compared to other racial or ethnic groups, will participate in this study. Children (age<18 years) are not eligible for this project.
Figure 3, and the flow diagram for the overall study design is shown in Figure 4. The study is currently being conducted at the University of Alabama at Birmingham (UAB) Clinical and Translational Science (CCTS) Clinical Research Unit, UAB Diabetes Research Center Human Physiology Core, and UAB Nutrition and Obesity Research Center Metabolism Core, where participants in intervention group are compensated $250 and participants in control group are compensated for $450 for their time. Participants are considered eligible for inclusion in this study if they meet the following criteria: 1) between the ages of 18 and 65 years; 2) diagnosis of traumatic SCI at the cervical, thoracic, or lumbar level (C5-L2) classified as American Spinal Injury Association (ASIA) Impairment Scale (AIS) A, B, C, or D; 3) impaired glucose tolerance or untreated type 2 diabetes; 4) no history of pre-existing self-reported type 2 diabetes and/or renal disease; and 5) at least 3 years post-injury (in our preliminary studies we have demonstrated that glucose intolerance develops as soon as 3 years post-injury). Interested participants undergo an oral glucose tolerance test (OGTT) to confirm eligibility (existing type 2 diabetes or impaired glucose tolerance). We comply with the American Diabetes Association 2016 recommendations (28) to identify individuals with SCI with either: type 2 diabetes (two-hour OGTT plasma glucose ≥200 mg/dL) or impaired glucose tolerance/pre-diabetes (two-hour OGTT plasma glucose 140 mg/dL to 199 mg/dL). Eligible participants are excluded if they develop new health conditions (e.g., pressure ulcers, kidney diseases, heart diseases, in need of hospitalizations) that would affect study outcomes or inhibit them from participating.
Recruitment
A two-stage procedure is employed to identify and recruit potential study participants. First, we use a computer-generated list of individuals with SCI who are enrolled in the UAB Spinal Cord Injury Model System (SCIMS). All potential participants are mailed a letter that describes the study, invites them to participate, and provides a return postcard indicating their willingness to participate. Responders are interviewed by phone to assess eligibility. Three months after the mailing, participants who have not responded are called in a final attempt at recruitment. If the necessary sample size is not achieved, a second stage of recruitment begins, which utilizes advertising at the community level, such as at the Lakeshore Foundation, a not-for-profit organization that has been helping people with disabilities for more than 15 years by providing education, recreation, and other support services. The second stage of recruitment also includes advertising at the UAB SCIMS website, newsletters and social media. Those who respond to the advertisement are evaluated against the study eligibility criteria. Eligible responders are recruited consecutively as study participants until the necessary sample size is achieved. The recruitment and enrollment of participants are performed by the study coordinator (JL) or by study staff under study coordinator’s direct supervision.
Interested participants are invited for screening visit. Study procedures, risks, and potential benefits are explained by the study coordinator or principle investigator (CYF) during the screening visit. Participants’ rights are stated in the IRB-approved consent form. Participants are given options to provide consent for collection and use of their data and specimens in ancillary studies.
Power Calculations
Power calculations were performed using nQuery Advisor + nTerim 3.0 and assumed a two-sided statistical test and a significance level of 5%. We obtained estimates of the standard deviation (SD) for two clinically important metrics of metabolic function: a Matsuda Index (indicator of insulin sensitivity) of 3.8 units (21), and 2-hour OGTT plasma glucose levels of 16 mg/dL(29). Given the dearth of publications related to the effects of diet composition on gut bacteria, we were unable to perform power calculations for gut bacteria composition; however, we have adequate power to detect statistically significant differences in primary metabolic outcomes.
As described in Figure 4, we intent to recruit 100 participants (50 per group) for the study, taking into account an estimated final participation of rate of 80% and an attrition rate of 20%. With a final sample size of 30 participants per group (LC/HP dietary intervention and control), and also assuming a two-group t-test and the prior assumptions, we have 80% power to detect between-group differences of 2.8 units in the Matsuda Index, and 11.8 mg/dL in 2-hour OGTT plasma glucose levels as being statistically significant. With this same sample size, and also assuming a paired t-test and the prior assumptions, we have 80% power to detect within-group differences of 2.1 units in the Matsuda Index and 8.5 mg/dL in 2-hour OGTT plasma glucose levels as being statistically significant. We believe that these estimates are conservative because we will be performing our primary statistical analyses for between-group and within-group comparisons simultaneously using statistical methods that are more sophisticated than those that are assumed here.
Interventions/Groups
LC/HP Dietary Intervention Group. The daily LC/HP dietary intervention includes ~30% total energy as protein (1.6 g/kg per day) with a carbohydrate-to-protein ratio <1.5 and fat intake set at ~30% of the total energy intake. These dietary parameters are designed to fall within the Acceptable Macronutrient Distribution Range established by the Institute of Medicine(30). The LC/HP dietary intervention meets the recommended daily intake for fiber, vitamins, and minerals for adults aged 18-60 years. Dietary fat sources focus on monounsaturated and polyunsaturated fats, e.g., plant oils and nuts; dietary carbohydrate sources emphasize whole grains, fruits, vegetables, and legumes; and dietary protein sources include lean meats, fish, chicken, eggs, and nonfat dairy foods, e.g., fat-free milk and low-fat cheese, consistent with American Diabetes Association and Institute of Medicine guidelines. All LC/HP meals are provided by UAB CCTS Bionutrition Unit and delivered to participants’ homes weekly (a sample menu is included in supplementary table 1, Additional file 2). Every delivery includes breakfast, lunch, dinner, and snacks for 7 days. Once a week during the study, participants are weighed by study personnel at their home with a portable wheel chair scale (Health O Meter 2400KL, 800 lbs capacity) to ensure weight stability. If weight changes exceed 2 kg from baseline, calorie modification is prescribed to maintain participants’ weight.
Overall energy/calorie needs of each participant is determined according to resting energy expenditure (REE), assessed via indirect calorimetry and multiplied by an activity factor. The Physical Activity Recall Assessment for People with Spinal Cord Injury (PARA-SCI)(31) is used to determine physical activity level and an appropriate activity factor (for estimation of calorie needs for each participant) for each subject. The PARA-SCI is an interviewer-administered recall questionnaire tailored exclusively to people with SCI who use a wheelchair as their primary mode of locomotion. PARA-SCI is just as reliable as measures that have been used in large-scale epidemiological studies of the general population (32). Physical activity levels may be different among groups (control and intervention) during the course of the study, which may mix in with the effects of diet and distort the true relationship of the intervention to outcomes in the intervention group. Therefore, we measure each participant’s’ physical activity levels via PARA-SCI(31) and physical activity levels will be treated as a potential confounder in our statistical analyses.
Resting Energy Expenditure (REE). Overall energy/calorie needs of each participant in the LC/HP diet group is determined according to REE. REE is measured at baseline (Week 1) after a 12-hour fast. Measurements are performed in a quiet and softly lit room. Temperature is maintained between 22ºC and 24ºC. Participants lay supine on a comfortable bed, with the head enclosed in a Plexiglas canopy. After resting for 15 minutes, REE is measured for 30 minutes with a computerized, open-circuit, indirect calorimetry system with a ventilated canopy (Vmax ENCORE 29N Systems, SensorMedics Corporation, Yorba Linda, CA). The last 20 minutes of measurement is used for analysis. Oxygen uptake (O2) and carbon dioxide production (CO2) is measured continuously and values are averaged at 1-minute intervals. REE is calculated from the O2 and CO2 data.
Safety of the LC/HP Dietary Intervention. Administering an LC/HP dietary intervention for a relatively short period of time (8 weeks) is not expected to produce any significant side effects. There have been no reported adverse side effects of this diet in long-term or short-term clinical trials in healthy or diabetic individuals (33-35). In fact, studies involving administration of an LC/HP diet for a relatively longer period of time (8-16 weeks) in obese and/or diabetic patients have shown improvements in metabolic and cardiovascular disease markers (visceral fat, blood glucose, and insulin levels)(36, 37).
Retention and Compliance. We expect voluntary attrition to be comparable to that of our diet and exercise training trials in the SCI population (20%). Participants who voluntarily withdraw prior to completing 8 weeks of study or data collection will be replaced with a new recruit on a per participant basis. Ensuring compliance with home consumption of the prescribed LC/HP diet may be challenging. In a prior study involving an LC/HP dietary intervention in individuals with SCI, Dr. Yarar-Fisher (PI) demonstrated nearly 100% compliance. In any event, subjects are given pre-configured meals with instructions to consume all prescribed foods. Dietary compliance is maximized via education with study coordinator, as well as involvement of the participant in individual food item selection from a menu of options (personalized menus). To improve both dietary and study retention, subjects assigned to the LC/HP dietary intervention group are contacted by phone 2 days per week to troubleshoot any potential barriers (food craving, poor appetite, constipation, loose or firm stool, etc.) to their progress in the study. A daily food checklist is provided to record adherence to the prescribed menu. Blood urea nitrogen (BUN) is measured before, at week 4, and 8 as a surrogate indicator of dietary protein intake during the study (38, 39).
Control Group. The control group does not receive any dietary intervention and are continuing with their regular daily diets. Participants complete three 24-hour food recalls (on 2 week days and one day in the weekend) three times (at weeks 1, 4 and 8) during the course of the study to gather dietary information including dietary intake and/or particular aspects of the diet. Participants are asked to recall foods and beverages they consumed in the 24 hours prior to the interview. Three 24-hour food recalls appear optimal for estimating energy intake (40, 41).
Outcome Measures and Analysis
Primary Outcomes
The primary outcomes are as follows:
- Metabolic function
- Glucose tolerance
- Insulin sensitivity
- β-cell function
- Lipid profile
- Body composition
- Total fat mass
- Total lean mass
- Visceral fat mass
- Gut microbiome
- Composition of gut bacteria
Clinical procedures to measure metabolic function.
Oral Glucose Tolerance Test (OGTT). Each subject consumes a 75-g oral glucose load within 5 minutes. Blood samples are collected immediately before and 10, 30, 60, 90, and 120 minutes after glucose ingestion for measurement of plasma glucose and plasma insulin. Blood for plasma glucose determination is collected with sodium fluoride and blood for plasma insulin is collected with heparin. Blood is immediately centrifuged and separated to obtain plasma, which is frozen at -80ºC until analysis. Assays are performed in the UAB Human Physiology and Metabolism Core. Plasma glucose, insulin, and C-peptide values are analyzed for measures of insulin sensitivity and β-cell (cells that control insulin secretion in pancreas) function using mathematical modelling techniques developed for data generated from the OGTT (37). Whole body insulin sensitivity (WBIS) also is calculated using the Matsuda Index (a formula based on insulin and glucose values measured during the OGTT) (42). Plasma glucose and C-peptide assays are performed on an automated analyzer (Sirrus analyzer; Stanbio Laboratory, Boerne, TX) and plasma insulin is measured using an immunofluorescent method with an AIA-600 II analyzer (TOSOH Bioscience, South San Francisco, CA) per the manufacturers’ instructions.
Lipid Analysis. Venous blood samples are collected to measure the participant’s total cholesterol, triglycerides (TG), HDL-C, and LDL-C levels in the fasting state. This blood is drawn using the catheter that is placed for the OGTT, so it does not require an extra needle stick. Total cholesterol, HDL-C, and TG are measured with the Sirrus analyzer. LDL-C levels are estimated using the formula LDL-C=total cholesterol − HDL-C − triacylglycerol/5 (reference PMID: 4337382). Analyses are performed in the UAB Human Physiology and Metabolism Core.
Clinical procedures to measure body composition
Dual-energy X-ray Absorptiometry (DXA). Total body imaging (to measure total fat and lean mass and visceral fat mass) is acquired using the GE Healthcare Lunar iDXA and analyzed using enCORE software version 13.6 in the UAB Human Physiology and Metabolism Core. Daily quality control scans are acquired during the study period. Participants are scanned using our standard imaging and positioning protocols (43, 44). For measuring visceral fat, a region of interest is automatically defined, with the caudal limit placed at the top of the iliac crest and the height set to 20% of the distance from the top of the iliac crest to the base of the skull to define the cephalad limit. Fat mass data from DXA are transformed into CT adipose tissue volume using a constant correction factor (0.94 g/cm3). This constant is generally consistent with the density of adipose tissue.
Procedures to measure changes in the composition of gut bacteria
Acquisition and Processing of Fecal Samples. Gut bacterial (microbial) composition patterns are determined from stool samples using our established protocol (45). Stool collection occurs at participants’ homes. Participants or their caregivers are provided with a pre-paid/labeled FedEx package containing a stool collection kit (Para-pak® vial for sample preservation, stool collection container, 2-gallon Ziploc bag for disposal of collection container, and sanitizing wipes). After bowel movement, approximately 5-10 mL of stool is transferred to the Para-pak® vial using the attached spatula in the Para-pak® vial. For participants who have their bowel management program on their beds, stool collection container are not used. Fedex packages containing stool samples are shipped to our lab using the overnight shipping option to ensure appropriate preservation of the microbiota. Upon delivery, the fecal samples are diluted in Cary-Blair medium to 0.1 mg/mL for a total volume of 20 mL with 10% by volume glycerol. Aliquots of 200 μL are stored at -80°C for DNA extraction/gut bacteria analysis. Fecal DNA is isolated via Fecal DNA Isolation Kit. The 16S rRNA gene amplification (metagenomics) protocol with the MiSeq System is used to characterize the composition of gut bacteria to the family level, and in some cases, the genus and species level (45). The relative abundance of identified organisms is normalized prior to analysis based on maximum read counts per sample. The organisms with low relative frequencies (<%0.1) are filtered. The remaining organisms are used for within-subject and between-subject comparison analyses.
Secondary Outcomes
The secondary outcomes are as follows:
- Quality of life
- Health related quality of life
Measurement of quality of life
Spinal Cord Injury-Quality of Life (SCI-QOL) Measurement System. Quality of life is measured via the SCI-QOL measurement system, which consists of a set of items that have been developed specifically for use in spinal cord medicine (46, 47). Three scales that measure physical and health-related quality of life are being used. Table 1.
Statistical Analysis
All data analyses will be carried out using data from participants that complete the study intervention. Descriptive statistics will be calculated for all study parameters of interest. Analyses for all aims include comparisons of means of outcome measures, which include the Matsuda Index (insulin sensitivity), 2-hour OGTT plasma glucose levels (glucose tolerance), β-cell function, total cholesterol, TG, LDL-C, and HDL-C, total lean mass, total fat mass, visceral fat mass, and composition of gut bacteria between the two groups (LC/HP diet, control diet) as well as comparisons within each group of the changes from the baseline visit to the Week 8 visit. The primary method of analysis is mixed models repeated measures analyses, such as repeated measures analysis of covariance. An appropriate structure for the covariance matrix (e.g., the unstructured covariance matrix) will be selected for these models using the final data. This method allows us to compare changes over time (8 weeks) and differences between groups simultaneously. Potential confounders, such as age, gender, injury level, time since injury, and levels of physical activity, will be accounted for in these analyses. Overall adjusted comparisons between groups at baseline will be performed using the two-group t-test, and overall unadjusted comparisons of the change from the baseline visit to the Week 8 visit is performed using the paired t-test. If assumptions of normality of distribution for the above tests are not tenable, variables may be log10 transformed prior to analysis or appropriate non-parametric tests such as the Wilcoxon rank-sum and signed-rank tests may be used. Generalized linear model techniques, such as regression analyses and mixed models repeated measures analyses, will be used to determine how changes in composition of gut bacteria are associated with improvements in metabolic function, or how improvements in metabolic function are related to improvements in quality of life. Secondary analysis of participants who have type 2 diabetes or glucose intolerance will be performed if the numbers of participants with these conditions differ greatly between the study groups. If we have missing data at study completion, we will not perform multiple imputation or apply the last observation carried forward method; instead, we will perform a secondary analysis of participants who completed the study vs. participants who did not complete the study and determine if these two groups differ in any substantial manner. Statistical tests will be two-sided and performed using a 5% significance level. SAS software, version 9.4 or later, will be used to conduct the statistical analyses.
Data management:
All study data are managed in a central database using REDCap, a secure web software system designed for clinical trials. All questionnaires are coded into REDCap and administered electronically. Data from outcome measures are output from the respective technical equipment in electronic or paper form and then uploaded directly into the REDCap database. However, data on adverse events that are collected by the research team are recorded on paper forms, stored in the Clinical Research Unit, and then manually entered into the REDCap database. Food intake data are captured on paper forms and double entered into the database.
Paper documents are scanned and saved on UAB's secure network and/or stored in locked cabinets in the research coordinator’s office. Data stored on UAB's secure network are protected through stringent security measures assured by UAB’s technical department and through the use of coded ID numbers and electronic security systems required by HIPAA. The REDCap software system is hosted locally on secure servers provided by the UAB School of Medicine. REDCap software leaves a pristine audit trail by documenting all changes to data, logging all user activity, and recording all pages viewed by every user. Moreover, it strictly controls access to data with password authentication and user privileges. This ensures that all users have limited access to only the data and information that they need or should have access to.
All data that are captured on paper forms and then manually entered into the database are verified by double entry (enter/verify). The REDCap database has logic checks built in to identify valid values, and any potentially erroneous data will be meticulously documented and triple-entered. The PI and study coordinator will periodically check the database for missing data and will document all such data and the reasons for absence. After the study is completed, all data will be quality checked by the statistician, PI, and the study coordinator in year 5; tasks will be divided among the three personnel according to their respective responsibilities. Statistical analyses for each endpoint will begin only after all data for that endpoint has been quality checked. In compliance with UAB’s IRB policies, all data and records will be kept for at least 3 years after the study is completed; all PHI for participants will be deleted 3 years after the trial is completed, while the de-identified final dataset will be retained indefinitely and published online for other scientists to benefit from.
Trial monitoring
All human participant data, ranging from recruitment/screening to diet intervention and testing to laboratory tests, are reviewed in quarterly (every 3 months) in Data and Safety Monitoring meetings attended by key study staff and the PI. This includes updates on any new hazards, risks, or adverse events, and plans of action. Additional investigators and staff will be asked to participate as the need for their input or expertise arises. If during the course of these meetings, particular unforeseen hazards or risks are identified that may predispose patients to an unusually high number of serious adverse events, the PI will consult the appropriate members of the investigative team, as well as the UAB IRB, to determine if the study should be terminated or altered in some way. Any procedure that is deemed hazardous will be eliminated from the study and replaced with an alternative if one with reasonable risk can be identified.
Adverse event monitoring and reporting
Participants are instructed to report adverse events as they occur. In case of emergency, participants are given a phone number to contact the study physician. Participants are given a separate phone number and email address to contact the study team to report non-urgent adverse events.
Documentation. All adverse events will be documented on specialized case report forms and graded on their attribution (unrelated to the protocol, or possibly, probably, or definitely related to the protocol), severity (mild, moderate, or severe), expectedness (unexpected versus expected), and frequency. We will also document any actions taken related to the adverse event and the outcome or resolution of the event.
Reporting. All serious or unexpected adverse events will be reported to the UAB IRB with a description of the event, when and how it was reported, and appropriate documentation to corroborate the event. The description of the adverse event will include all information listed on the case report forms. The IRB will then determine whether additional reporting to NIDILRR is required. All adverse events will be reported immediately to both the study physician and PI, and then, in turn, reported to the IRB within 10 business days.
Protocol Amendments
Any change to the protocol will require a written protocol amendment that must be approved by NIDILRR and IRB before implementation. Upon acceptance from the sponsor and IRB, the PI will make updates and edits to the study record published on ClinicalTrials.gov. If the PI determines that an immediate change to or deviation from the protocol is necessary for safety reasons to eliminate an immediate hazard to the subjects, the IRB will be notified immediately.
Confidentiality
To protect privacy, each study participant is assigned a unique 3-digit identification number that cannot be traced to any protected health information (PHI). The PI and study coordinator code all data forms, participant information, and biological specimens using these ID numbers. No PHI appears on these materials; instead, the keys linking participants’ identities to their unique identification numbers are being stored separately in a passcode-secured storage disk. To protect privacy and confidentiality, any original paperwork documenting a participant’s name and PHI are stored in a locked cabinet in the research coordinator's office, while any digital study records involving PHI are stored in REDCap and/or on computers requiring password authentication that are stored in locked offices and that are behind secure firewalls. Paperwork involving PHI are kept to the bare minimum necessary and are stored in a locked cabinet in the research coordinator's office. Thereafter, each generated data and specimen are labeled with a unique anonymous ID number.
Access to data
Only study staff, the University of Alabama at Birmingham (UAB) Institutional Review Board (IRB), official overseers of clinical research at UAB, and representatives of the NIDILRR have access to study records, data, and specimens; all access is on a need-to-know basis. Study staff include all key personnel, the dietician, the study coordinator, the study statistician, and any other individuals who perform specific tests or procedures for the study, such as nurses and lab technicians. All study staff are trained in HIPAA standards for protecting PHI and will not refer to PHI or confidential information in the presence of individuals outside of the study team. Moreover, each study staff member's access to participants’ data is limited to only the functions for which s/he is responsible. Records that identify study participants are kept confidential as required by law, and every effort are being made to maintain the confidentiality of participants’ study records. Except when required by law or if necessary to protect their rights or welfare, study participants are not identified by name or any other identifying characteristic in records disclosed to those outside of the study staff.
Dissemination Policy.
We will disseminate our results in several ways: 1) Journal publications. We will publish our results in peer-reviewed journals with open access policies and/or in top-tier journals. 2) Scientific meetings. We will present our results at scientific conferences, including at the American Congress of Rehabilitation Medicine annual meeting, the American Spinal Injury Association annual meeting, and the Experimental Biology annual meeting. 3) Electronic Media. We will publish our findings on a) The UAB Spinal Cord Injury Model System Information Network Website. The website features a comprehensive collection of links (over 360 to date) to SCI-related information provided by reputable organizations, associations and educational institutions. All educational materials written and produced from this project will be made available free on this website. b) Pushin’on eNewsletter by the UAB SCIMS. The newsletter provides persons with SCI and their families with information of interest. Over the years, the newsletter has featured original articles along with news, information, and synopses of research of importance to individuals with SCI. A synopsis of the proposed research trial, including a description of the diet-intervention and a lay summary of major findings and how to implement them will be published on the newsletter.