2.7. Neopterin Measurement by HPLC

Neopterin levels were assayed in the urine by high-performance liquid chromatography (HPLC). Urine samples were collected following these two criteria: (i) collect urine without any direct intervention to avoid stress; or (ii) obtain pure urine without contamination with feces or animal feed. The changes in urinary neopterin levels depend on the degree of peripheral immune system activation, which is also directly connected to the intensity of exercise, duration and training status. Samples of urine were collected at 0 and 1, 3, 7, 10 and 14 days after initiation of the study. Samples were collected by the single animal method described previously. Briefly, an individual mouse urinates on plastic wrap, outside of the cage [34]. The urine is then collected into a microcentrifuge tube, quickly spun down and frozen at −80 °C. When all samples are collected, they are centrifuged at 16,000× g for 10 min at 4 °C and diluted in 10 volumes (v/v) of 15 mM potassium phosphate buffer, containing 5 mM EDTA. The HPLC analysis of urinary neopterin was determined using a Supercosil LC-18-T 5 μm reverse-phase column (15 × 4.6 mm), using a flow rate of 0.7 mL/min with an elution of 85% 15 mM potassium phosphate buffer, containing 15% acetonitrile, pH 6.4. The column temperature was maintained at 35 °C. The neopterin was identified and quantified by a multi-wavelength fluorescence detector (module 2475, Waters, Milford, MA, USA) with an excitation wavelength of 355 nm and an emission wavelength of 438 nm [35]. The results were expressed as μmol (neopterin)/mol creatinine. Creatinine levels were analyzed using a colorimetric kit (ThermoFisher, Waltham, MA, USA).