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

Abstract

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-HPLC 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.

Pterins as diagnostic markers of exercise-induced stress: a systematic review

“…32 Smith et al 32 measured no change up to 6 h post-cycling at 140 beat/min for one hour suggesting the intensity was not sufficient to induce T-cell release of ␥-IFN. In contrast, cycling 230 km, 35 4763 km, 33 to exhaustion 36,37 or repetitively at maximal intensity for 30 s 34 all resulted in significant increases that remained elevated from 60 min 36 to 16 days. 33 Total neopterin also increased immediately post-cycling in response to a continuous exhaustion protocol 36 or repeated maximal bouts of 30 s. 34 When biopterins were measured, cycling at 80% VO 2 max caused a 150% increase in BH 4 and total biopterin 38 while repeated maximal bouts of 30 s 34 only increased biopterin.…”

“…Second, their concentrations can be measured in several bio-fluids including plasma, serum, CSF, saliva and urine, the latter two of which allows for a non-invasive and stress free collection while CSF can provide quantitative information on several brain cortices associated with injury. Third, pterins are easily measured by commercially available equipment including ELISA, RIA 55 and using reverse phase, 56 cation exchange 50 or amino 36 high-performance liquid chromatography. Because of their natural fluorescence and high absorbance, pterins can be measured in concentrations in the nM range.…”

A simple and useful method for evaluation of oxidative stress in vivo by spectrofluorometric estimation of urinary pteridines

“…In our preliminary experiments, the fluorescence intensities of kynurenine at an excitation wavelength of 360 nm and emission wavelengths from 400 to 540 nm were much lower (less than 1%) than those of neopterin and biopterin. Moreover, a transient oxidative burst induced by a short-term exhaustion exercise has recently been reported to result in a significant increase in neopterin, but not kynurenine, in blood 26 . Therefore, kynurenine may not contribute to oxidative stress level estimated by fluorometrical measurement in the present study.…”

Oxidative stress and immune cell activation quantification in sepsis and non-sepsis critical care patients by neopterin/7,8-dihydroneopterin analysis

“…It may be possible for bias to be introduced when only assessing one body fluid, particularly with the effect of kidney function discussed below. Moreover, 7,8-dihydroneopterin has been shown to be more stable when in urine than plasma [33,38]. Total neopterin over neopterin (TNP/NP) ratios were determined for sepsis and non-sepsis patients.…”