Non Technical Summary
Cottonseed oil (CSO) has been shown to improve blood lipids in healthy and at-risk adults. CSO is a rich source of poly-unsaturated fatty acids (PUFAs). These positive effects on lipid metabolism may be due to the general fatty acid (FA) composition of CSO; however, our evidence shows that a FA unique to CSO, dihydrosterculic acid (DHSA), may be responsible. It is unknown in humans whether a FA-matched diet to that of CSO would yield similar effects on lipid metabolism. This would elucidate whether it is the FA composition in general, or whether CSO (and DHSA) is required to observe improvements in lipid metabolism. Our study goal is to compare CSO to a FA composition-matched diet ("PUFA") on changes in fasting and postprandial lipid metabolism and chronic disease risk factors. Aims: (1) Examine the impact of CSO vs. PUFA on fasting and postprandial lipids, and (2) Examine the impact of CSO vs. PUFA on other markers of chronic disease risk (markers of glycemia, inflammation, and lipid peroxidation).We will employ a randomized control trial with three arms: a CSO-enriched diet (CSO) group, a mixed oil diet matching the FA composition of CSO (PUFA) group, and a control. The protocol includes six study visits and a 28-day partial outpatient feeding protocol. If our hypotheses are correct, this study could have an enormous impact on the cotton industry by demonstrating the superior health benefits of CSO consumption.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	1810	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
1810 - Cottonseed;

Field Of Science
1010 - Nutrition and metabolism;

Goals / Objectives
Cottonseed oil (CSO) has been shown to improve blood lipids in healthy and at-risk adults. CSO is a rich source of poly-unsaturated fatty acids (PUFAs). These positive effects on lipid metabolism may be due to the general fatty acid (FA) composition of CSO; however, our evidence shows that a FA unique to CSO, dihydrosterculic acid (DHSA), may be responsible. It is unknown in humans whether a FA-matched diet to that of CSO would yield similar effects on lipid metabolism. This would elucidate whether it is the FA composition in general, or whether CSO (and DHSA) is required to observe improvements in lipid metabolism.Our goal is to compare CSO to a FA composition-matched diet on changes in fasting and postprandial lipid metabolism and markers of chronic disease risk.Our global hypothesis is that the CSO-enriched diet will improve fasting and postprandial lipid and chronic disease risk markers more than a diet of similar FA composition.Specific Aim/Supporting Objective 1: Examine the impact of CSO vs. PUFA on lipid metabolism in two ways: Assess fasting blood lipids Hypothesis: CSO will result in improved blood lipids (namely lower TC, LDL-c, and TGs and higher HDL-c), and these changes will be better than PUFA, with no change in CON (CSO>PUFA>CON).Approach: Measure fasting blood lipids and lipid subfractions pre- and post-diet.Assess postprandial plasma markers of lipid metabolismHypothesis: Postprandial TGs and NEFA will be reduced to the greatest degree with CSO, followed by PUFA, and no change in CON (CSO>PUFA>CON).Approach: 5h postprandial blood draws following a high SFA meal challenge will be used at pre- and post-diet intervention.Specific Aim/Supporting Objective 2: Examine the impact of CSO vs. PUFA on other markers of chronic disease riskHypothesis: Inflammatory markers (TNF-α, IL-6, CRP, and IL-1β) will be unchanged at post-intervention and not different between groups. Glycemic markers (glucose, insulin, HOMA-IR, HOMA-B) and lipid peroxidation (malondialdehyde (MDA)) will improve in CSO vs. both PUFA and CON.Approach: Fasting and postprandial blood draws for glycemia and MDA, and fasting blood draws for inflammation, will be compared at pre- and post-diet.

Project Methods
Study Design: This will be a randomized, double-blinded, control trial with 3 groups: an intervention group on a CSO-enriched diet (CSO, n=30), a mixed oil diet matching the FA composition (same % total of PUFA, MUFA, SFA) of CSO (PUFA, n=30), and a control group (CON, n=30) that will consume a mixed oil diet containing similar FA distribution (MUFA, PUFA, SFA) to that of the U.S. diet.Participants: Ninety adults (n=30 per group) will be recruited to participate. Inclusion criteria include men and women between the ages of 30-70y who are at-risk for CVD. Participants who meet the inclusion criteria will be screened prior to starting the study. Exclusion criteria will include weight change >5% of body weight in the past 3mo., regular exercise (>3h/wk), history of medical or surgical event that could affect digestion or swallowing, gastrointestinal surgeries, conditions or disorders, history of previous or current renal or bowel disease, women on hormone replacement therapy for <2 years, chronic diseases (e.g. cancer, diabetes), medications affecting digestion, absorption, or metabolism (e.g. thyroid meds), lipid-lowering medications, diabetes medications, steroid/ hormone therapies, current antibiotics, pregnancy or nursing, or tobacco/nicotine users. Individuals who are on a medically prescribed or special diet or have food allergies (specific for the foods in the study), those taking fish oil, or excessive alcohol use will also be excluded. This study will be approved by the IRB, and informed written consent will be obtained from each participant prior to testing.Protocol:Screening Visit (v0): Participants will report to the HNL following an overnight fast (8-12h) and no exercise or alcohol for at least 24h. Anthropometrics including height, weight, blood pressure (BP), and hip & waist circumference will be measured. BMI will be calculated to confirm eligibility if blood lipid criteria are not met. Body composition will be measured using Dual Energy X-Ray Absorptiometry. Resting metabolic rate (RMR) will be measured for 30 minutes on the TrueOne 2400 (Parvo Medics, Sandy, UT) following standard procedures. The final 20 minutes of data collection will be used to calculate RMR using the full Weir equation 101. Estimated total daily energy needs to determine the amount of intervention goods and HF meal challenge at visits 1 and 5 will be based on a participant's RMR*1.65. A fasting blood sample will be obtained to assess standard blood lipids and fasting blood glucose. To be included in the study, participants must be "at cardiovascular risk" as defined by either an unfavorable blood lipid profile or BMI classification of overweight or greater (≥25kg/m2). Additionally, fasting blood glucose must be < 126 mg/dL to be eligible.Lead-in Procedures: If eligible, participants will be scheduled for the baseline, pre-diet intervention visit (v1). Subjects will complete a 3d food diary (two week days and one weekend day) to assess baseline dietary intake. The night before v1, participants will be provided with a lead-in dinner and a snack that contains 50% or total energy from carbohydrate, 15% from protein, and 35% from fat.Pre-Diet Intervention Visit (v1): Participants will arrive at the HNL in a fasted state (8-12h fast) and 24h without exercise or alcohol. Researchers will confirm that the lead-in dinner meal and snack were consumed the night before. Subjects will also complete the International Physical Activity Questionnaire (IPAQ) 103 to assess physical activity during the last 7 days. Anthropometric measurements (described in screening visit) will be taken, an IV catheter will be inserted for intermittent blood draws, and a fasting sample will be obtained to measure blood lipids and other markers of chronic disease risk. Participants will then complete a HF, SFA-rich meal challenge. The meal is comprised of Ensure, chocolate powder, oils (palm and coconut oil) and butter. The participant will have 5min to consume the meal. The nutrient content of the meal will be designed to provide 25% of the participant's total estimated daily energy needs. After ingestion, participants will fill out a sensory questionnaire 104 based on the meal. After ingestion of the meal, blood samples will be drawn at 30, 60, 90, 120, 150, 180, 240 and 300min postprandially) for analysis of blood lipids and other markers of chronic disease risk.28-Day Diet Intervention: Following v1, participants will begin the 28-day diet intervention. Participants will be given a one-week supply of daily shakes and snacks that corresponded to their assigned diet group. The shake and snack will be designed to provide 20% of their total daily energy needs from CSO, PUFA, or CON oils. Participants will be instructed to consume the shake for breakfast and the snack during the day while following their usual diet for the remainder of the day. The snack will be provided on a rotating menu of yogurt or pudding that are enriched with CSO, PUFA, or CON.All food items will be weighed and prepared by research personnel in our research kitchen (which includes 1 registered dietitian and 3 dietetic interns) with all ingredients weighed to the 0.01g. For shake consumption, participants will be instructed to prepare this liquid meal by mixing provided individually portioned instant meal shake mix, with milk of choice, and a designated amount of the assigned oil, also provided. The snacks will be pre-weighed and individually packaged for each day's consumption.The CSO treatment will have CSO as the only added oil. The PUFA treatment will contain a mixture of 3 oils (corn, safflower, and coconut) and is designed to match the FA composition/saturation of the CSO diet. The PUFA diet contains 54.0% n-6 and 0.09% n-3 PUFA, which closely matches CSO (53.7% n-6 and 0.13% n-3 PUFA). The CON diet will contain a mixture of 3 oils (corn, canola, and coconut) and will provide a FA composition that is equal to that reported in the U.S. diet. The PUFA breakdown will be 23.1% n-6 and 2.4% n-3 PUFA. By providing the same foods (but with different oils) we will ensure that participants are consuming the same items with the only difference being the type of oils added to the foods.During weeks 2 and 4 of the intervention, participants will complete 3d food diaries). During the entire intervention, there will be a meal compliance list to write down the time each food item is consumed. To be included in final analysis for this intent to treat design, compliance will be set at 80%, which is standard practice across dietary intervention studies 106,107. All participants will be given sensory questionnaires 104 to fill out once/week for the breakfast shake and snack to assess palatability and acceptability.During Intervention Visits (v2-v4): The 28d intervention will begin the day after v1. Participants will consume the breakfast shake and snack daily and follow their usual diet for the remainder of the day. The IPAQ and 3-day food diaries will be completed during weeks 2 and 4. Participants will return to the lab once a week (day 7 (v2), day 14 (v3), and day 21 (v4)) for a short study visit. At these visits, a fasting blood draw and body weight will be collected. Participants will also consume their breakfast shake in the lab, turn in study documents, and collect their meals for the next 7 days. Those documents include (1) meal compliance log (and saved food containers to return to lab), (2) 3d food diaries and IPAQ (v3 only), and the (3) sensory evaluation log.Post-Diet Intervention Visit (v5): The day following the 28th day of the intervention period, participants will report to the HNL under fasting and un-exercised conditions as described previously. They will consume the same lead-in meal and snack as the night before v1. All procedures that take place at v1 will be repeated. Participants will also turn in their meal compliance log and a 3d food diary from the last 7 days.