Table 4 Brain health, cardiometabolic biomarkers and more comprehensive methodology descriptions

Glucose

Spectrophotometric-hexokinase method using the Abbott Alinity Analyser

Insulin

Chemiluminescent Microparticle Immunoassay using the Abbott Alinity Analyser

Total cholesterol

Standard enzymatic methods using the Abbott Alinity Analyser

HDL

Accelerator Selective Detergent using the Abbott Alinity Analyser

LDL

LDL will be calculated using the Friedewald formula using the Abbott Alinity Analyser

Hs-CRP

Immunioturbidimetric method using the Abbott Alinity Analyser

HbA1c

Boranate Affinity HPLC method using the Trinity Premier Hb9210.

Full blood examination (FBE)

Beckman Coulter DXH800 instrument

TNF-α, IL-6, IL-1B, adiponectin, and leptin, BDNF,

MAGPIX multiplex microsphere-based immunoassay (Millipore, Billerica, MA)

IGF-1

Human IGF-I/IGF-1 Quantikine ELISA (R&D Systems)

Apolipoprotein E 4 (ApoE-4)

Stored in PAXgene DNA tube, PAXgene Blood DNA kits (QIAGEN) used for DNA isolation, PCR will be performed using custom design primers for the amplification of a segment of the ApoE gene and the 96-well Taqman Gene Expression qPCR Assay (Thermofisher Scientific). ApoE Sequencing (of the PCR product will occur using two single nucleotide polymorphisms (SNPs). Validation of sequencing results will be completed by targeted genotyping via differential amplification of ε2, ε3, or ε3 alleles with custom designed primer pairs.

Serum and plasma analyses

In preparation for serum analyses, samples will be collected in SST tubes and, for plasma analysis, in EDTA tubes. Both samples are rested for thirty minutes in the fridge, prior to centrifuging at 2000 RPM for 15 min at 4 degrees, with the separated plasma and serum aliquoted into 400ul and 750ul volumes and stored at −80 degrees celcius.