RESUMO
Fibrosing chronic graft-versus-host disease (cGVHD) is a debilitating complication of allogeneic stem cell transplantation (alloSCT). A driver of fibrosis is the kynurenine (Kyn) pathway, and Kyn metabolism patterns and cytokines may influence cGVHD severity and manifestation (fibrosing versus gastrointestinal [GI] cGVHD). Using a liquid chromatography-tandem mass spectrometry approach on sera obtained from 425 patients with allografts, we identified high CXCL9, high indoleamine-2,3-dioxygenase (IDO) activity, and an activated Kyn pathway as common characteristics in all cGVHD subtypes. Specific Kyn metabolism patterns could be identified for non-severe cGVHD, severe GI cGVHD, and fibrosing cGVHD, respectively. Specifically, fibrosing cGVHD was associated with a distinct pathway shift toward anthranilic and kynurenic acid, correlating with reduced activity of the vitamin-B2-dependent kynurenine monooxygenase, low vitamin B6, and increased interleukin-18. The Kyn metabolite signature is a candidate biomarker for severe fibrosing cGVHD and provides a rationale for translational trials on prophylactic vitamin B2/B6 supplementation for cGVHD prevention.
Assuntos
Doença Enxerto-Hospedeiro/sangue , Ácido Cinurênico/sangue , Cinurenina/sangue , Riboflavina/sangue , Transplante de Células-Tronco , Vitamina B 6/sangue , Adolescente , Adulto , Idoso , Quimiocina CXCL9/sangue , Quimiocina CXCL9/genética , Feminino , Fibrose , Regulação da Expressão Gênica , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/sangue , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Interleucina-18/sangue , Interleucina-18/genética , Quinurenina 3-Mono-Oxigenase/sangue , Quinurenina 3-Mono-Oxigenase/genética , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Leucemia/terapia , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Linfoma/terapia , Masculino , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Estudos Retrospectivos , Índice de Gravidade de Doença , Transdução de Sinais , Transplante Homólogo , Triptofano/sangue , ortoaminobenzoatos/sangueRESUMO
This work describes a simple, sensitive and rapid liquid chromatography-high resolution mass spectrometry method for the quantitation of perhexiline and the simultaneous detection of perhexiline metabolites in C57bl/6 mice plasma. Only 5 µL of plasma was used for analysis. Pretreatment was limited to a 100-fold dilution ('dilute-and-shoot'). The analyte was detected by high resolution mass spectrometry (Orbitrap™ technology). Three scan events were performed over the entire chromatogram. Targeted single ion monitoring with data dependent acquisition was employed for perhexiline quantitation and confirmation, while full scan was used to perform untargeted detection of perhexiline phase I and phase II circulating metabolites. The calibration curve was linear (r(2)=0.990) ranging from 0.305 ng/mL (LLOQ) to 10000 ng/mL. Matrix effect was limited to 6.1%. The method was applied to a pharmacokinetic study of perhexiline in mouse plasma and the results obtained were compared to a standard sample preparation method based on protein precipitation and liquid chromatography-tandem mass spectrometry (MRM mode) detection. The new approach provided comparable results in terms of pharmacokinetics parameters estimate with a high sensitivity, additional information on perhexiline circulating metabolites and a low consumption of biological sample. The combination of the 'dilute-and-shoot' approach together with HRMS targeted and untargeted detection represents a suitable alternative to classic bioanalytical approaches in preclinical research.