Plasma and urinary NMR metabolic profiling of the phenylalanine-tyrosine pathway in nitisinone-treated alkaptonuria: comparison of two dosing regimens and identification of a candidate pharmacodynamic biomarker

Alkaptonuria is an ultra-rare autosomal recessive inborn error of metabolism caused by a deficiency of homogentisate 1,2-dioxygenase, leading to the systemic accumulation of homogentisate and progressive ochronotic deposition in connective tissues. Nitisinone, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase, effectively reduces homogentisate production but causes secondary hyper-tyrosinaemia, posing a risk of ocular complications. This study employed 1H NMR spectroscopy to characterise the metabolic profiles of plasma and urine samples collected from 12 patients with alkaptonuria before and after three months of nitisinone treatment. Quantification of key intermediates in the tyrosine catabolism pathway revealed a marked reduction in urinary homogentisate, confirming drug efficacy, alongside a substantial rise in plasma tyrosine levels. Urinary metabolites upstream of 4-hydroxyphenylpyruvate dioxygenase showed significant accumulation, consistent with an enzymatic block. Comparison of two dosage regimens (10 mg once daily vs 10 mg on every other day) identified 4-hydroxyphenylpyruvate as the only metabolite exhibiting a statistically significant inter-regimen difference, while plasma tyrosine remained elevated in both groups. These findings highlight the complementary diagnostic value of plasma and urinary NMR profiling and underscore the limitations of current dosage strategies in controlling nitisinone-induced hyper-tyrosinaemia. NMR metabolomics provides a non-invasive, simultaneous snapshot of pathway-wide metabolic changes and represents a powerful tool for personalised therapeutic monitoring in AKU.