Targeted LC-MS/MS metabolomics reveals distinctive tryptophan pathway signatures in pneumocystis jirovecii pneumonia

Pneumocystis jirovecii pneumonia (PJP) is a high-mortality fungal infectious disease, and the changes in tryptophan metabolism and its diagnostic value in PJP remain unclear. This study aimed to elucidate the specific tryptophan metabolic profile in PJP patients by establishing and validating an LC-MS/MS method for the simultaneous quantification of tryptophan and its nine key metabolites in human serum. Serum samples from 46 PJP patients, 35 pulmonary tuberculosis (PTB) patients and 40 healthy controls were analyzed. The method achieved complete chromatographic separation within 5.5 min, with an extraction recovery rate > 95.8 % and matrix effects ranging from 86.0 % to 106.8 %, meeting the comprehensive validation criteria set by FDA and EMA guidelines. PJP patients exhibited significantly higher serum levels of 3-hydroxykynurenine (3−HK) compared to the PTB and healthy control groups (26.0 vs. 11.5 vs. 8.94 ng/mL, p  < 0.001), while levels of 5-hydroxytryptophan and indole-3-acetic acid were significantly lower ( p  < 0.05). The 3-Hydroxyanthranilic Acid/3-hydroxykynurenine ratio was also markedly reduced in the PJP group. ROC analysis demonstrated strong diagnostic performance for PJP with 3-hydroxykynurenine (AUC = 0.919), 5-hydroxytryptophan (AUC = 0.816), indole-3-acetic acid (AUC = 0.705), and the 3-Hydroxyanthranilic Acid/3-hydroxykynurenine ratio (AUC = 0.743). The validated LC-MS/MS method is rapid, sensitive, high-throughput and enables precise analysis of tryptophan metabolism. The distinct metabolic signature—particularly elevated 3-hydroxykynurenine, reduced 5-hydroxytryptophan and indole-3-acetic acid, and an altered 3-Hydroxyanthranilic Acid/3-hydroxykynurenine ratio—provides novel biomarkers for PJP diagnosis and offers new insights into its immunometabolic mechanisms.