# Urinary Tryptophan–Kynurenine Pathway Profiling in Bulgarian Children with Autism Spectrum Disorder (ASD): Neopterin Co-Varies with Kynurenine and Quinolinic Acid

## TL;DR

This study found that in children with autism in Bulgaria, certain urine chemicals linked to immune and brain function are connected, suggesting possible biological patterns in autism.

## Contribution

The study identifies novel associations between neopterin and kynurenine pathway metabolites in a pediatric ASD cohort.

## Key findings

- Neopterin levels in urine strongly correlate with quinolinic acid and kynurenine in children with ASD.
- These associations remain significant even after adjusting for creatinine levels.
- The findings suggest a potential link between immune activity and tryptophan metabolism in ASD.

## Abstract

Background/Objectives: Autism spectrum disorder (ASD) is biologically heterogeneous, and immune-linked variation may be associated with differences in tryptophan–kynurenine pathway (KP) metabolism. Here, we report a targeted urinary profile of KP metabolites, NAD (nicotinamide adenine dinucleotide), and neopterin in a Bulgarian pediatric ASD cohort to describe within-cohort patterns and associations. Methods: Second-morning, acid-stabilized spot urine was collected from 73 children with ASD in Bulgaria (3–13 years; 57 males; 16 females). No contemporaneous neurotypical control group was enrolled; therefore, laboratory-provided reference limits are reported only as contextual benchmarks and are not interpreted as ASD-specific abnormalities. Tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), NAD, and neopterin were quantified and derived indices were computed (KYN/TRP × 1000; QUIN/KYNA). Non-parametric statistics, Benjamini–Hochberg false discovery rate (FDR) correction, and Spearman correlation analyses were applied. Results: Neopterin was strongly associated with QUIN and KYN in creatinine-normalized data (QUIN: ρ = 0.59, q36 = 2.64 × 10−7; KYN: ρ = 0.54, q36 = 3.69 × 10−6); these associations persisted when reconstructed as absolute concentrations (e.g., QUIN_abs: ρ = 0.68, q36 = 2.69 × 10−10) and after partial Spearman correlation controlling for spot creatinine (partial ρ = 0.46, q = 2.52 × 10−4). One NAD value was <LOQ and was imputed as ½LOQ; sensitivity analyses did not materially change inference. Conclusions: In this ASD-only cross-sectional dataset, urinary neopterin levels co-varied with urinary KYN and QUIN and with KP indices. Clinical interpretation and causal inference require controlled and longitudinal studies with richer covariate capture.