Clinical evaluation of plasma neopterin as a biomarker of immune activation in allergic rhinitis using a fluorescence-based o-phthaldehyde derivatization method

Abstract

Allergic rhinitis (AR) is a common chronic inflammatory condition of the nasal mucosa driven by IgE-mediated hypersensitivity, yet reliable biomarkers of systemic immune activity and oxidative stress remain limited despite involvement of Th2 cytokines, eosinophils, and macrophages. Neopterin (NPT) is a macrophage-derived marker of interferon-γ-induced immune activation and oxidative imbalance, but current plasma detection methods are expensive, or lack sufficient sensitivity for routine clinical use. Herein, a validated fluorescence-based method was developed for determination of plasma NPT using o-phthaldehyde derivatization in the presence of 2-mercaptoethanol under mildly alkaline conditions. The reaction formed a stable isoindole derivative with strong fluorescence (λ_ex = 335 nm, λ_em = 455 nm), increasing the quantum yield from 0.24 for native NPT to 0.65 for the NPT-OPA product. The method showed linearity from 1 to 20 ng/mL (r² = 0.9996), with a detection limit of 0.22 ng/mL and a quantification limit of 0.66 ng/mL. Accuracy ranged from 99.0% to 100.8%, and precision remained below 1.0%. No interference appeared from biopterin, folic acid, riboflavin, or xanthopterin, each contributing less than 5% of the NPT signal. Plasma NPT was measured in 26 AR patients and 26 healthy controls. AR patients showed elevated NPT concentrations (2.9 ± 0.7 ng/mL) compared to controls (1.5 ± 0.3 ng/mL, p < 0.001). NPT levels correlated with symptom severity (r = 0.44, p = 0.015) and eosinophil counts (r = 0.41, p = 0.022), indicating an association between immune activation and disease intensity. The fluorescence-based method provides a sensitive, selective, and rapid approach for plasma NPT quantification and supports NPT as a potential biomarker of immune activation and oxidative stress in AR.