Differential Absorption Kinetics of Grape Seed Extract Polyphenols

Grape seed extracts (GSEs) are widely studied for their potential cardiometabolic health benefits, largely due to their high (poly)phenol content. GSEs can differ substantially in their (poly)phenol composition, particularly in their degree of polymerization, which may lead to differences in absorption, metabolism, and biological efficacy. GSE (poly)phenols include compounds ranging from monomers, such as catechin and epicatechin, to oligomeric and polymeric compounds. Monomeric forms are absorbed in the small intestine, whereas oligomeric and polymeric forms have limited direct absorption and may undergo microbial metabolism in the colon, producing smaller metabolites that can be absorbed systemically. This pilot study will evaluate the absorption and clearance kinetics and metabolite profiles of GSEs with varying degrees of polymerization in healthy adults.

STUDY DESIGN

This study consists of two separate pilot studies using a randomized, double-blinded, crossover design.Study 1 will compare MN-BP with another commercially available GSE. Study 2 will evaluate next-generation (Gen 2) GSE preparations enriched in monomers, oligomers, polymers, or a mixture. Participants will receive different GSE interventions during separate intervention periods according to a randomized sequence. A washout period will occur between interventions to minimize potential carryover effects. Both studies will follow similar study procedures; the GSE interventions will differ.

SCREENING VISIT

Participants eligibility for the study will be determined based on the study inclusion and exclusion criteria. During the Screening Visit, the following general measurements will be obtained: height, weight, body composition, waist circumference, blood pressure, heart rate, and body temperature. Fasting blood glucose will be measured using a finger prick. A pregnancy test will also be performed when applicable. Participants will complete demographic, health, and medication questionnaires. They will be asked to provide information about their demographics, health and medical history, medications, dietary habits, and other information relevant to determining study eligibility. Based on the results of the fasting finger-prick blood glucose, questionnaires, physical measurements, and evaluation of general health, participants who meet the eligibility criteria may be invited to continue in the study.

PRE-STUDY VISIT

The Pre-Study Visit may be performed on the same day as the Screening Visit. This visit will last approximately 45 minutes to 1 hour. Study staff will provide detailed instructions about the study procedures and how participants should prepare for each study day. Participants will be trained on study procedures, including foods to avoid, medications and supplements to avoid, food diary recording and overnight fasting. Study dates will be scheduled, and participants will receive a study booklet containing the study calendar and study instructions. Participants will also be provided with a frozen dinner to consume the day before the next intervention visit. If participants are taking medications or supplements that do not interfere with the study or have been approved by the study staff, they will continue taking them as instructed by their healthcare provider and record them in their diary. Study staff will provide instructions regarding foods and supplements that are allowed or should be avoided during the study.

STUDY DAY VISITS

At the beginning of each intervention visit, study staff will collect study materials from the participant, including the participant's diary, and provide the 24-hour dietary recall (ASA24). Study staff will confirm that the participant has followed the required study-day preparation instructions. Fasting blood glucose will be checked by finger prick to verify usual fasting glucose. Before beginning the intervention, anthropometric measurements, including weight, waist circumference, and body composition, will be obtained. Vital signs, including blood pressure, heart rate, and body temperature, will also be measured.

GSE ADMINISTRATION AND BLOOD COLLECTION

Participants will meet with a Licensed Health Care Provider for blood sample collection. An intravenous catheter (IV) will be placed in the participant's arm or hand to allow multiple blood samples to be collected during the study visit while minimizing repeated needle sticks. If there is difficulty obtaining blood through the IV catheter, participants may be asked whether they are comfortable with individual blood collections using a butterfly needle. There will be eight blood draws over approximately 8hours during the intervention visit. Immediately after catheter placement and collection of the first blood sample, participants will receive the assigned GSE treatment to take with water. The next blood sample will be collected approximately 1 hour later.Serial blood samples will be collected throughout the study period to assess GSE-derived parent (poly)phenolic compounds and their metabolites and characterize their absorption over time.

MEALS DURING THE STUDY VISIT

Participants will receive standardized low-(poly)phenol meals during the intervention visit. Breakfast will be provided during the morning portion of the study visit, and a low-(poly)phenol lunch will also be provided during the visit.After completion of the scheduled blood and urine collections, participants will be provided with a dinner meal to take home.

URINE COLLECTION

Urine samples will be collected at multiple time points according to the study schedule. Participants will be provided with water during the study visit, and the amount of water consumed will be recorded. Blood and urine samples will subsequently be used to characterize parent (poly)phenolic compounds and their metabolites and evaluate the absorption, clearance, bioavailability, and metabolic profiles of the GSE interventions.

24-HOUR FOLLOW-UP VISIT

After completing the intervention-day procedures and consuming the provided dinner, participants will return to the clinic the following day in a fasting state, consuming only water during the fasting period. At the follow-up visit, a fasting blood sample and urine sample will be collected after taking fasting glucose through finger prick and blood pressure.

These samples will provide the later time-point measurements needed to evaluate the absorption and clearance of GSE-derived (poly)phenolic compounds and their metabolites.

WASHOUT AND CROSSOVER

Following completion of one intervention period, participants will undergo a washout period of 7-10 days before the next intervention to minimize potential carryover effects. Participants will then repeat the required dietary preparation and study-day procedures before receiving the next GSE intervention according to their randomized crossover sequence.

The intervention procedures-including pre-study dietary restrictions, anthropometric and vital-sign measurements, GSE administration, serial blood collection, standardized low-(poly)phenol meals, urine collection, and the fasting follow-up visit-will be repeated for the subsequent GSE interventions.

SAMPLE ANALYSIS

Venous blood will be collected in EDTA tubes, and plasma will be separated. Blood and urine samples will be stored at -80°C until analysis. GSE-derived (poly)phenols and their metabolites will be characterized in the collected plasma and urine samples. The samples will be analyzed using ultra-high-performance liquid chromatography and mass spectrometry (UHPLC-MS).

The resulting concentration-time profiles will be used to evaluate absorption and clearance kinetics, bioavailability, and metabolic profiles of the different GSE preparations.