Apolipoprotein E (APOE) Genotype Effects on Triglycerides and Blood Flow in the Human Brain

Overview: The purpose of the study is to enroll 90 older adults, half of whom will have E4 carrier status, to assess whether E4 status affects change in global cerebral blood flow (∆CBF) in response to a high fat drink. The screening visit includes informed consent, baseline blood draw, oral triglyceride tolerance test (OTTT), and dual-energy x-ray absorptiometry (DEXA). The study visit involves assessing cerebral blood flow using arterial spin labeling (ASL) MRI - at baseline and at 2 hours after drinking heavy cream. The study visit will also include 6 blood draws and a 30 minute cognitive battery.

Screening Visit Procedures (at Seattle South Lake Union campus): Before the screening visit, participants will receive a copy of the consent form in the mail as well as a medical history questionnaire to fill out. Participants will be advised to fast the night before the screening visit (minimum 8 hours required). After an informed consent process, study personnel will check vitals, including waist circumference, height, and weight for body mass index (BMI) calculation. An IV will be placed by the study nurse and blood sent for complete blood count, chem20 (including liver and kidney function assessment), glycated hemoglobin (hemoglobin A1C), lipid panel, and a blood sample for APOE genotyping (Northwest Lipids using polymerase chain reaction (PCR) methodology). Blood samples except APOE will be sent to the University of Washington (UW) Clinical Laboratory Improvement Amendment (CLIA)-certified lab, and participants will receive a copy of the results by mail. After the blood draw, participants will then undergo an oral glucose tolerance test (OGTT): 6 time points (0, 15, 30, 45, 60,120 min) which will include a blood draw for plasma to measure insulin and C peptide (in batches by Northwest (NW) lipids). At time 0 and 120 minutes study personnel will also assess vitals and a point of care glucose using a glucometer. The OGTT will be the standard 75 grams glucose drink from central supply. After the 2-hour OGTT is complete, patients will undergo a DEXA scan. Note, DEXA can be performed before OGTT if needed for study flow. Parking will be provided, and participants will be paid with a gift card.

Screening visit sample day:

8 am: Check in to the clinic 8-8:30: informed consent in private room, go over medical history 8:30-9: Insert IV, fill out depression, anxiety screen, draw baseline blood 9 -11 am: OGTT (During gaps in OGTT, do Montreal Cognitive Assessment (MoCA)) 11-11:25 am: DEXA 11:25-11:30: Remove IV, distribute gift card and parking validation Time: 3.5 hours, total blood drawn 40 mls

Study Visit Procedures (at University of Washington main campus): Participants will arrive to the UW Translational Research Unit (TRU) after an overnight fast (minimum 8 hours required). The study nurse will place an IV, and check vital signs including blood pressure, heart rate, temperature, and weight. Then, the participant will be escorted to the MRI scanner and undergo the fasting imaging protocol. The participant will head back to the TRU and start the oral triglyceride tolerance test (OTTT). Time 0 blood is drawn and time documented. Then, the participant will drink the dairy product-100 mLs of heavy cream (just under half a cup) which contains 370 calories, 40.4 grams of total fat, 23.6 grams of saturated fat. The participant will be encouraged to drink the product over 5 minutes as per published OTTT protocols. Blood draws will be collected for measurement of glucose, insulin, lipids and appetite hormones (30, 120, 180, 240 min). A post-lipid MRI will be done at the 2 hour time point, and the NIH toolbox cognitive assessment will be done at 3 hour time point. The NIH battery takes approximately 30 minutes to administer and will be done on a portable tablet.

Study visit sample day:

8 am: Check in to UW TRU, ask about new medications, place IV 8:15: Escort down to MRI area, get pre-MRI 8:45: Back at TRU: T0 blood draw 8:45-8:50: Drink lipid 9:20: T30 min blood draw 9:50: T60 min blood draw 10:50: T120 min (2 hour) blood draw

Return to MRI scanner for 2 hour MRI (will be ~2 hours and 10 minutes post drink) 11:50: T180 min (3 hour) blood draw Do the NIH toolbox testing now - takes about 30 minutes 12:50: T240 min (4 hour) blood draw 12:50-1 pm: Remove IV, receipt of gift card, participant checks out. Time: 5 hours, total blood drawn: 80 mls (13 per blood draw x 6 time points)

MRI protocol: The investigator will use pseudo-continuous arterial spin labeling to measure cerebral blood flow (CBF) in ml/100g/min as a marker of perfusion for improved signal quality. In this approach, magnetically labeled arterial blood water serves as the endogenous contrast. MRI images will be stored on external hard drive with encryption and will be uploaded to One Drive or another HIPAA-compliant system for further processing and analysis.

Experiment: As per the consensus recommendations, the investigator will use a sequence (5.5 minutes) with long label duration = 1.8 s, long post-labeling delay = 2 s, with labeling offset = 25-30 mm, slices = 20, spatial resolution = 3.5×3.5×5 mm3, field of view = 240×240×100 mm3, SENSE-factor = 2, repetition time/echo time (TR/TE)= 5000/18 ms. Dual adiabatic background suppression pulses will be applied to minimize gray and white matter tissue contamination at inversion delay (TI) = 2.05 and 3.25 s. Finally, the investigator will acquire an equilibrium magnetization scan (M0, 1 minute), identical to the above scan, but with TR = 10,000 ms and no labeling or background suppression.

Analysis: First, motion correction will be applied to the arterial spin labeling images using motion correction (FSL-MCFLIRT), and these will be registered to the M0 image. Then, the investigator will perform a pair-wise subtraction between the control and null images and apply a two-compartment model to quantify CBF. The resulting CBF map will be co-registered to the T1 scan followed by a transformation to Montreal Neurological Institute (MNI) space.The temporal regions such as the entorhinal cortex, temporal lobe, hippocampus as well as the posterior cingulate and lateral parietal lobules will be identified using standard Harvard-Oxford Cortical and Subcortical atlas. CBF values will be compared in these regions between the groups. The region of interest approach can dilute effects that are smaller than the size of the region of interest. Therefore, the investigator will also perform permutations testing in FSL to evaluate voxel-wise CBF differences between group. In order to ensure that the voxel-wise outcomes are not mere chance, the investigator will impose strict family-wise error correction for multiple comparisons.

Data collected for this study:

Data collected on the phone and on medical history form (verbal consent given, prior to informed consent):

Medical history, including if participants know their APOE genotype Targeted review of systems Age of menopause (for women) Medications Use of drugs, tobacco, and alcohol Drug allergies Food allergies/intolerances Handedness Work and Education history of participant Education level of mother (if known) Education level of father (if known) MRI checklist: surgeries, metal in body

Data collected at screening visit:

How patient identifies as to race/ethnicity Vitals: Height, weight, waist circumference measures Physical Health Questionnaire-9 (PHQ9, a depression screen) Generalized Anxiety Disorder-7 Assessment (GAD-7, an anxiety screen) Cognitive assessment: MoCA version 8.2 English version Screening bloodwork: chemistries, hepatic function, lipid panel, hemoglobin A1C, Complete Blood Count (CBC), APOE genotype (cheek swab and blood test) Fasting and 2 hour point of care glucose (using a glucometer) Fasting and 2 hour heart rate, blood pressure Six OGTT blood draws: 4 ml lavender top tube to measure glucose, insulin (sent to lab in batches) DEXA scan for standard body composition measures (i.e. fat mass, fat-free mass)

Data collected at study visit:

Vitals: Heart Rate (HR), BP, Respiratory Rate (RR), temperature at all time points Six blood draws: serum and plasma collection for glucose, insulin, lipids and appetite hormones NIH toolbox cognitive data MRI ASL data: Total and regional ASL images (does not include a clinical MRI read)