2.3. Skeletal muscle nitrate and nitrite analysis

Skeletal Muscle samples were collected from the gastrocnemius muscle using a Bergstrom needle with suction (on a separate day to the maximal treadmill exercise test) as previously reported (Woessner et al., 2017). All muscle samples were kept frozen until analysis of muscle nitrate and nitrite (Park, Thomas, Wylie, et al., 2021). Muscle samples were initially weighed (20–150 mg wet weight), mixed with nitrite preserving solution (K₃Fe(CN)₆, N‐ethylmaleimide, water, Nonidet P‐40), as described in (Park, Thomas, Wylie, et al., 2021) and homogenized using a GentleMacs homogenizer (Miltenyi Biotec Inc). Proteins were then precipitated using cold methanol. Specifically, thawed samples were mixed with ice‐cold methanol (dilution 1:3 sample: methanol) and then centrifuged at 11,000 g for 5 min at 4°C. Supernatants were collected and immediately used to determine nitrate and nitrite content by chemiluminescence. The nitrite and nitrate content were determined by a Sievers gas‐phase chemiluminescence NO analyzer (Sievers 280i Nitric Oxide Analyzer, GE Analytical Instruments). Nitrate and nitrite data are presented as nanomoles per gram wet weight of tissue.

Following baseline testing, participants were randomly assigned to consume either 70 ml (4.2 mmol NO₃ ⁻) beetroot (EX+BR) or an identical nitrate‐depleted placebo (EX+PL), 2.5 hours before each exercise training visit (3× per week for 12 weeks), as previously detailed (Woessner, VanBruggen, et al., 2018). Each training session included at least 30 min of actual walking, with the intensity tailored to each participant's initial baseline maximal graded exercise test results. When claudication pain became moderately severe (3–4/5 on a 5‐point claudication pain scale), they would step off the treadmill and rest until the pain subsided (rest periods were not included in the 30‐min exercise time). Typically, after a patient was able to walk 8 to 10 min at the initial workload, the grade was increased by 0.5%, or the speed increased by 0.1 mph as tolerated.

Each exercise testing visits included, on separate days a 6 min walk distance (6MWD) test and maximal treadmill exercise test with a 12‐lead ECG to measure peak walk time (PWT) and claudication onset time (COT) (Woessner et al., 2017).