Metabolomic studies in the inborn error of metabolism alkaptonuria reveal new biotransformations in tyrosine metabolism

Abstract

Alkaptonuria (AKU) is an inherited disorder of tyrosine metabolism caused by lack of active enzyme homogentisate 1,2-dioxygenase (HGD). The primary consequence of HGD deficiency is increased circulating homogentisic acid (HGA), the main agent in the pathology of AKU disease. Here we report the first metabolomic analysis of AKU homozygous Hgd knockout (Hgd ^-/-) mice to model the wider metabolic effects of Hgd deletion and the implication for AKU in humans. Untargeted metabolic profiling was performed on urine from Hgd ^-/- AKU (n = 15) and Hgd ^+/- non-AKU control (n = 14) mice by liquid chromatography high-resolution time-of-flight mass spectrometry (Experiment 1). The metabolites showing alteration in Hgd ^-/- were further investigated in AKU mice (n = 18) and patients from the UK National AKU Centre (n = 25) at baseline and after treatment with the HGA-lowering agent nitisinone (Experiment 2). A metabolic flux experiment was carried out after administration of ¹³C-labelled HGA to Hgd ^-/-(n = 4) and Hgd ^+/-(n = 4) mice (Experiment 3) to confirm direct association with HGA. Hgd ^-/- mice showed the expected increase in HGA, together with unexpected alterations in tyrosine, purine and TCA-cycle pathways. Metabolites with the greatest abundance increases in Hgd ^-/- were HGA and previously unreported sulfate and glucuronide HGA conjugates, these were decreased in mice and patients on nitisinone and shown to be products from HGA by the ¹³C-labelled HGA tracer. Our findings reveal that increased HGA in AKU undergoes further metabolism by mainly phase II biotransformations. The data advance our understanding of overall tyrosine metabolism, demonstrating how specific metabolic conditions can elucidate hitherto undiscovered pathways in biochemistry and metabolism.

Keywords: AKU, alkaptonuria; AMRT, accurate mass/retention time; Alkaptonuria; Biotransformation; CV, coefficient of variation; FC, fold change; FDR, false-discovery rate; HGA, homogentisic acid; HGD, homogentisate 1,2-dioxygenase; HPPD, hydroxyphenylpyruvic acid dioxygenase; LC-QTOF-MS, liquid chromatography quadrupole time-of-flight mass spectrometry; MS/MS, tandem mass spectrometry; MSC, Molecular Structure Correlator; Metabolism; Metabolomics; Mice; PCA, principal component analysis; QC, quality control; RT, retention time.

Figure 1

Schematic overview of the overall study design, incorporating Experiments 1–3. In Experiment 1, urine was collected from Hgd^−/− and Hgd^+/− mice and profiled by LC-QTOF-MS. Targeted and non-targeted feature extraction was performed on the data in parallel and subsequent unpaired t-tests were employed to identify differentially abundant compounds between Hgd^−/− and Hgd^+/−. These compounds were then further investigated in LC-QTOF-MS data from two additional datasets; a previously published study examining the effect of nitisinone on the urine metabolome of Hgd^−/− BALB/c mice and patients with AKU (Experiment 2) and a plasma flux analysis using a ¹³C₆ labelled HGA tracer (Experiment 3).

Figure 2

Clear differences between the urine metabolomes of Hgd^−/− and Hgd^+/− mice. (A–D) PCA on data from targeted feature extraction, with PCA plots showing separation between Hgd^−/− and Hgd^+/− mice by component 1 in A, negative, and B, positive ionisation polarities. Lower plots show the corresponding PCA loadings of metabolites on components 1 and 2 in C, negative, and D, positive polarity. (E, F) Volcano plots illustrating selection of statistically significant urinary metabolites between Hgd^−/− and Hgd^+/− mice based on p-value and fold change. (E) negative polarity; (F) positive polarity. Compounds with P < 0.05 (Benjamini-Hochberg FDR adjusted) and log₂ fold change >1.5 are labelled, with red and blue indicating increased and decreased abundance, respectively, in Hgd^−/−. Turquoise indicates adjusted P < 0.05 but log₂ fold change <1.5. Bold text indicates that the increase observed in Hgd^−/− was confirmed in mouse plasma following injection with ¹³C₆ HGA tracer. ∗ Compound not previously reported in the literature.

Figure 3

Isotopologue extraction results on plasma from the in vivo metabolic flux experiment using injected ¹³C₆-labelled homogentisic acid (HGA). Data shown are from Hgd^−/− and Hgd^+/− samples taken at intervals of 2, 5, 10, 20, 40 and 60 (when possible) min after injection. Extracted ion chromatograms (EIC’s) represent the M+0 (native compound) and M+6 (¹³C₆-labelled form) isotopologue signals for HGA, HGA-sulfate and HGA-glucuronide. EIC’s show clear M+6 peaks for these compounds following injection (but only from Hgd^−/− mice for HGA-glucuronide), confirming that they are derived from the labelled HGA.

Figure 4

Predicted structures of newly-identified HGA biotransformation products resulting from phase I and II metabolism. Predicted structures were the closest matches against the acquired experimental MS/MS data, based on scores obtained using Agilent Molecular Structure Correlator (MSC). The proposed sites for metabolism/conjugation were based on match scores obtained for a list of possible candidates using a combination of MSC and CFM-ID 3.0in silico fragmentation modelling tools.

Figure 5

Summary of metabolites altered in Hgd^−/− mouse urine grouped by their associated pathways. Left: the tyrosine degradation pathway showing lack of the enzyme HGD in AKU and the consequential increase in HGA. Right (boxes): observed metabolite alterations grouped by pathway; red and blue indicate increased and decreased abundance respectively. Tyrosine metabolites, including HGA and HGA biotransformation products, were elevated. Metabolites associated with the TCA cycle were decreased. A combination of increased and decreased abundance was observed for purine pathway metabolites.