Abstract
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.
Highlights
IntroductionMeasurement is typically conducted by performance liquid chromatography; Indoleamine hC1ig8ho-rpSeCrfXorcmolaunmcensl.iHquoiwdevcherr,okmynatuorgernaipnheyan(Hd PtrLyCp)touppLsheainatirngFAd, 2eA,n3∗-odotiefodxayiaggeeonlndasaeal;inzidmabmleleut nFVe-ladincetenivaoarttemionaa.pvseoctnorVs,peaacceh over F with of which acts nite p on an are not measured simultaneously to neopterin uirsriendgucible tridiagonal fashion
The optimal mobile phase conditions for the separation and detection of neopterin, tryptophan and kynurenine in a single HPLC run were determined to be 28% 10 mM ammonium acetate with 0.3% formic acid and 72% acetonitrile
We investigated the stability of both 7,8-dihydroneopterin and neopterin in human plasma samples (Fig. 3). 7,8-Dihydroneopterin was oxidized for measurement as total neopterin and shows a significant decrease over 12 hours, with the largest loss being at 37°C
Summary
Measurement is typically conducted by performance liquid chromatography; Indoleamine hC1ig8ho-rpSeCrfXorcmolaunmcensl.iHquoiwdevcherr,okmynatuorgernaipnheyan(Hd PtrLyCp)touppLsheainatirngFAd, 2eA,n3∗-odotiefodxayiaggeeonlndasaeal;inzidmabmleleut nFVe-ladincetenivaoarttemionaa.pvseoctnorVs,peaacceh over F with of which acts nite p on an are not measured simultaneously to neopterin uirsriendgucible tridiagonal fashion. Such a pair is called a Leonard pair We have used an amA,inAe∗ isInsatirdotodbuecsteilfo-dnual whenever there exists an automorphism of the column for separation and simultaneous determinatisown aopf s A and A∗. In this case such an automorphism is unique, and called neopterin, kynurenine and tryptophan.
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