Simultaneous analysis of neopterin, kynurenine and tryptophan by amine-HPLC shows minor oxidative stress from short-term exhaustion exercise

Introduction: Neopterin, kynurenine and tryptophan can be used to measure activation of monocytes and macrophages during immunological events such as exercise inducing inflammation. Endurance exercise and high-impact sports have shown significant increases in these biomarkers. Measurement is typically conducted by high-performance liquid chromatography (HPLC) using C18 or SCX columns. However, kynurenine and tryptophan are not measured simultaneously to neopterin using these separation systems. Here we have used an amine column for separation and simultaneous determination of neopterin, kynurenine and tryptophan. Methods: Optimization and validation for the amine-IIPLC method was conducted using plasma from 43 participants subjected to a short maximal exercise bicycling regime or rest period. The order of exercise and rest was randomized and separated by a 3-5 week washout period. Results: Using an amine column developed with ammonium acetate formic acid (33%) and acetonitrile (72%) provided optimal separation and run time for analysis. Neopterin increased significantly post-exercise and subsided to baseline by 30 minutes. Total neopterin remained elevated until 60 minutes following exercise. Conclusion: Amine-HPLC can be used for simultaneous determination of kynurenine, tryptophan and neopterin in plasma. Short intense exercise causes a significant increase in plasma neopterin suggesting a prolonged activation of monocytes and macrophages.