Effect of Caffeine Consumption on Protein Biomarkers and Cardiovascular Disease

Participants would take two Wake-Ups 200mg Caffeine tablets to meet the target dose of 400mg of caffeine. These are a registered natural health product with Health Canada (NPN 80003474). They are developed by Adrem Brands, which was established in 1951. Quality control testing is performed at a third-party independent lab, Nucro-Technics, which is audited by Health Canada and the US FDA. This ensures that the caffeine tablets comply with Health Canada standards. The tablets contain caffeine anhydrous-a processed, dehydrated form of caffeine-which allows for a standard dose per tablet. The tablets are sugar-free, which reduces the confounding effect of sugar on our results; thus, the investigators can be more confident that the obtained results are reflective of the association between caffeine and the CVD-related protein biomarkers.

Participants would take two placebo pills to match the dosage of the intervention group. The investigators will use empty vegetable capsules from Health Bloom as the placebo for the control group. These capsules contain no contaminants or additives and are free from the common allergens. They are sugar-free-unlike most placebo pills, which reduces the confounding effect of sugar on our results. They are developed by Health Bloom, which is a Canadian company established in 2015. These capsules undergo testing at various stages of production to ensure purity and quality control. Each ingredient is tested prior to formulation, and the final capsules are subjected to thorough testing post-development. The capsules are white, and similar in appearance to the caffeine pill given to participants in the intervention group.

TASSO+ is a blood lancet device that uses negative pressure to draw capillary blood from the upper arm into a standard SST tube of volume 500μL. The small volume of blood collected makes this device less invasive than traditional collection methods. It is also safer and easier to use since it offers automated sample collection. TASSO manufactures a number of special devices for capillary blood sampling that have been validated as safe for consumer use, and the Tasso+ device is licensed with Health Canada for research use.

The Whatman Protein 903 Saver Card is a specialized filter paper designed for the collection, storage, and elution of biological samples, including blood. It contains five half-inch circles that hold 75 to 80 µL of blood, making it an efficient and reliable method of obtaining dried blood spot (DBS) samples. The participant's finger will be pricked using a sterile, disposable lancet, and drops of blood will be collected on the Whatman 903 Protein Saver Card to fill nine ½ inch discs via capillary action. The blood spots will be allowed to dry at ambient temperature (21°C) for two hours. Whatman Protein 903 Saver Cards undergo strict quality control and GMP manufacturing standards, which ensures high quality and reproducibility.

Blood will be collected from the medial cubital vein according to the standard venous blood sampling protocol into an EDTA tube with a volume up to 4mL.