Vacutainers system

In accordance with standardised guidelines [43 (link)] and after 15 min of seated rest, an automated sphygmomanometer (Omron, Omron Healthcare, UK) measured resting blood pressure on the brachial artery of the bare right arm two times, at one minute intervals. If the difference between the two measures was ≥5 mmHg, a third measure was taken and the mean calculated. A 15 ml fasting blood sample was taken from the antecubital vein of one arm using standard venepuncture technique (Vacutainers Systems, Becton-Dickinson, USA). Samples were collected into vacutainers containing edetate disociom or lithium heparin, immediately labelled with the unique participant number, and stored on ice during transportation to University laboratories for later analysis of glucose, total cholesterol and triglycerides.

Blood samples were obtained from the antecubital vein of the forearm via standard venepuncture technique (Vacutainers Systems, Becton–Dickinson). Samples were collected into vacutainers containing Silica (Clot Activator) and stored on ice until centrifugation for 10-min at 1,200 g at 4 °C. Serum aliquots were stored at − 80 °C for subsequent analyses. Commercially available pre-coated high-sensitivity ELISA kits (Thermo Fisher Scientific) were used to determine interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α) and high-sensitivity c-reactive protein (hs-CRP), while commercially available pre-coated standard ELISA kits (Thermo Fisher Scientific) were used to determine tissue plasminogen activator (t-PA) and von Willebrand factor (vWF). IL-6, TNF-α and hs-CRP were analysed as they are implicated in mood disorders [21 (link)] and cognitive functioning [22 (link)]. t-PA and vWF were analysed owing to their association with reductions in CBF [23 (link)]. Kits were stored and utilised according to the manufacturer’s instructions. An automated plate reader (CLARIOstar, BMG LABTECH GmbH, Offenburg, Germany) was used to read the raw absorbance values at the 450 nm wavelength for all assays. Each sample was analysed in duplicate.

Fasting blood samples were obtained from the antecubital vein of one arm via standard venepuncture technique (Vacutainers Systems, Becton-Dickinson). Samples were collected into vacutainers containing EDTA or lithium heparin and stored on ice until centrifugation for 15 min at 1500 g at 4 °C. Plasma aliquots were stored at −80 °C for subsequent analysis. Plasma glucose, triglycerides and total cholesterol concentrations were determined spectrophotometrically using commercially available kits (Randox Laboratories, Antrim, UK). Each sample was analysed in duplicate.