Insights into the role of diet and dietary flavanols in cognitive aging: results of a randomized controlled trial

With the world's population aging, age-related memory decline is an impending cognitive epidemic. Assessing the impact of diet on cognitive aging, we conducted a controlled, randomized, parallel-arm dietary intervention with 211 healthy adults (50–75 years) investigating effects of either a placebo or 260, 510 and 770 mg/day of cocoa flavanols for 12-weeks followed by 8-weeks washout. The primary outcome was a newly-developed object-recognition task localized to the hippocampus’ dentate gyrus. Secondary outcomes included a hippocampal-dependent list-learning task and a prefrontal cortex-dependent list-sorting task. The alternative Healthy Eating Index and a biomarker of flavanol intake (gVLM) were measured. In an MRI substudy, hippocampal cerebral blood volume was mapped. Object-recognition and list-sorting performance did not correlate with baseline diet quality and did not improve after flavanol intake. However, the hippocampal-dependent list-learning performance was directly associated with baseline diet quality and improved after flavanol intake, particularly in participants in the bottom tertile of baseline diet quality. In the imaging substudy, a region-of-interest analysis was negative but a voxel-based-analysis suggested that dietary flavanols target the dentate gyrus. While replication is needed, these findings suggest that diet in general, and dietary flavanols in particular, may be associated with memory function of the aging hippocampus and normal cognitive decline.


SUPPLEMENTARY FIGURE 1
Supplementary Figure 1: Changes in CBV-fMRI (not adjusted for baseline) before and 12 weeks after daily intake of placebo and flavanols at a low (260 mg), medium (510 mg) and high (770 mg) intake level. Data are expressed as mean ± SD (not adjusted for baseline).

NEUROIMAGING
Exclusion Criteria MRI-related exclusion criteria included cardiac pacemaker, internal pump, insulin pump, tattoo eyeliner, wire suture, internal metal objects, metal slivers in eye, prosthesis, hearing aid implants, neurostimulator, metal fragments, brain aneurysm clips, vascular clips, breast expander, vena cava filter, heart valve, metal stents, asthmatic symptoms within the past 3 years, sickle cell disease, kidney disease, pregnant, claustrophobic, wheelchair bound, machinist or ever worked with heavy metals and contraindication to gadolinium.

MRI Acquisition
Subjects eligible for MRI scans received them at two times (weeks 0 and 12) on a GE Prisma 3.0T MRI scanner using similar acquisition criteria as previously published 1,2 . Eligibility for MRI scanning included eligibility criteria for injection of Dotarem contrast agent. To be eligible for scanning, all subjects must not have received any contrast injection prior and undergo a fingerstick creatinine measure to measure glomerular filtration rate (GFR) the day of the scanner which falls below 60ml/min. Briefly, subjects were under the supervision of a physician or nurse who placed an intravenous line and loaded an appropriate weight adjusted dose of Dotarem contrast agent (0.1 mg/kg) into an auto-injector. A structural TFE T1-weighted high resolution (1x1x1 mm 3 ) structural image was acquired for cortical and subcortical segmentation. A structural T2 GRE image was acquired in addition to aid in the hippocampal segmentation. Following that, a pair of TFE T1 weighted images were acquired (0.68x0.68x3mm 3 ) oblique to the long axis of the hippocampus, prior to and after the bolus injection of the contrast agent and a four-minute period of rest. Patients remained in the scanner during the entire procedure.

MRI Processing
Whole brain CBV images were generated from the pair of pre and post-contrast MRI images as described previously 1 . In short, co-registered pre-contrast images were subtracted from postcontrast images and divided by a value of pure blood signal obtained from an automatic segmentation of the superior sagittal sinus. Structural images were analyzed through FreeSurfer 6.0 3 using both T1 and T2 images. The hippocampal subfield segmentation algorithm 4 was applied to images with a trained rater editing images. The CA4-DG-ML regions were merged for each participant's hippocampus bilaterally to capture the region of the DG, and further restricted from the 3 rd slice anteriorly, and 4 th slice posteriorly to localize the body of the hippocampus. Weighted mean values for all DG body ROIs were obtained after a filter was previously applied to exclude the effects of epicortical and high signal reflective of pure vasculature.

TEST MATERIAL DESCRIPTION
All test materials were provided by Mars, Inc. The vegetarian capsules either contained a placebo or varying levels of cocoa extract (Cocoapro®-processed cocoa extract) formulated to deliver 0 mg, 260 mg, 510 mg or 770 mg of cocoa flavanols per 4-capsule serving. The total amount of cocoa flavanols refrenced here is defined as the sum of all monomeric flavanols and their oligomers (i.e., procyanidins) with a degree of polymerization up to and including 7 (i.e. DP 1-7). The total cocoa flavanol content and flavanol stereoisomers were determined by HPLC with fluorescence detection in accordance with Bussy et al. 1 and Machonis et al. 2 , respectively. Placebo capsules contained biologically inert materials. Detailed information of the test material composition are provided in Table A. All capsules were indistingusiable in appearance. Capsules were provided in 120-count (30-d supply) bottles, labeled with an alphanumeric code. All participants and all researchers involved in the execution of the study remined masked with regard to the designation of the test material until post-study database lock and formal unmasking.