Quantification of the flux of tyrosine pathway metabolites during nitisinone treatment of Alkaptonuria

Abstract

Nitisinone decreases homogentisic acid (HGA) in Alkaptonuria (AKU) by inhibiting the tyrosine metabolic pathway in humans. The effect of different daily doses of nitisinone on circulating and 24 h urinary excretion of phenylalanine (PA), tyrosine (TYR), hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and HGA in patients with AKU was studied over a four week period. Forty AKU patients, randomised into five groups of eight patients, received doses of 1, 2, 4 or 8 mg of nitisinone daily, or no drug (control). Metabolites were analysed by tandem mass spectrometry in 24 h urine and serum samples collected before and after nitisinone. Serum metabolites were corrected for total body water and the sum of 24 hr urine plus total body water metabolites of PA, TYR, HPPA, HPLA and HGA were determined. Body weight and urine urea were used to check on stability of diet and metabolism over the 4 weeks of study. The sum of quantities of urine metabolites (PA, TYR, HPPA, HPLA and HGA) were similar pre- and post-nitisinone. The sum of total body water metabolites were significantly higher post-nitisinone (p < 0.0001) at all doses. Similarly, combined 24 hr urine:total body water ratios for all analytes were significantly higher post-nitisinone, compared with pre-nitisinone baseline for all doses (p = 0.0002 - p < 0.0001). Significantly higher concentrations of metabolites from the tyrosine metabolic pathway were observed in a dose dependant manner following treatment with nitisinone and we speculate that, for the first time, experimental evidence of the metabolite pool that would otherwise be directed towards pigment formation, has been unmasked.

Trial registration: ClinicalTrials.gov NCT01828463.

Conflict of interest statement

Milan, Hughes, Davison, Khedr, Rovensky, Psarelli, Cox, Rhodes, Gallagher and Ranganath report that they have no competing interests (financial or non-financial).

Figures

Figure 1

24-hour profiles of serum TYR [Mean(SD)] concentrations in SONIA 1 at baseline (a) and following treatment for 4 weeks with 0, 1, 2, 4 and 8 mg of nitisinone. (b) (Note y axis are different scales).

Figure 2

Metabolites pre and post nitisinone. Panel A shows sum of metabolites in 24 hour urine pre- and post nitisinone at doses of 0, 1, 2, 4 and 8 mg daily (µmol/24 hr). Panel B shows sum of total body water metabolites pre- and post nitisinone at doses of 0, 1, 2, 4 and 8 mg daily (µmol). Panel C shows sum of metabolites in 24-hour urine plus total body water metabolites pre- and post nitisinone at doses of 0, 1, 2, 4 and 8 mg daily. Values expressed as boxplots (25–75%) with interquartile range (5 and 95%).

Figure 3

Schematic of the effects of nitisinone on HGA. HGA accumulates in AKU due to a deficiency of homogentisate dioxygenase, with spill over into urine and also accumulation in the body as pigment. In AKU (Panel A), circulating HGA is in low concentration relative to urinary excretion and therefore the urinary excretion is dominant. However, data post-nitisinone shows that approximately 55% of TYR flows towards pigment formation and only 45% is excreted in the urine, following 8 mg nitisinone daily for 4 weeks. Panel B shows the complete inhibition of HGA excretion and HGA deposition and the suggested mechanism for metabolite generation post high dose nitisinone treatment. (4HPPD: 4-hydroxyphenylpyruvate dioxygenase; PA: phenylalanine; TYR: tyrosine; HPPA: 4-hydroxyphenylpyruvate; HPLA: 4-hydroxyphenyllactate; HGA: homogentisic acid; MAA: maleylacetoacetic acid). Kidney image is provided free of copyright under Creative Commons CC0 1.0 licence from Pixabay; permission is provided without attribution.