Evaluation of the serum metabolome of patients with alkaptonuria before and after two years of treatment with nitisinone using LC‐QTOF‐MS

Abstract

Background The homogentisic acid‐lowering therapy nitisinone is being evaluated for the treatment of alkaptonuria (AKU) at the National Centre for AKU. Beyond hypertyrosinemia, the wider metabolic consequences of its use are largely unknown. The aim of this work was to evaluate the impact of nitisinone on the serum metabolome of patients with AKU after 12 and 24 months of treatment. Methods Deproteinized serum from 25 patients with AKU (mean age[±SD] 51.1 ± 14.9 years, 12 male) was analyzed using the 1290 Infinity II liquid chromatography system coupled to a 6550 quadrupole time‐of‐flight mass spectrometry (Agilent, UK). Raw data were processed using a batch targeted feature extraction algorithm and an accurate mass retention time database containing 469 intermediary metabolites (MW 72‐785). Matched entities (±10 ppm theoretical accurate mass and ±0.3 minutes retention time window) were filtered based on their frequency and variability (<25% CV) in group quality control samples, and repeated measures statistical significance analysis with Benjamini‐Hochberg false discovery rate adjustment was used to assess changes in metabolite abundance. Results Eight metabolites increased in abundance (log 2 fold change [FC] 2.1‐15.2, P < .05); 7 of 8 entities were related to tyrosine metabolism, and 13 decreased in abundance (log 2 FC 1.5‐15.5, P < .05); including entities related to tyrosine (n = 2), tryptophan (n = 3), xanthine (n = 2), and citric acid cycle metabolism (n = 2). Conclusions Evaluation of the serum metabolome of patients with AKU showed a significant difference in the abundance of several metabolites following treatment with nitisinone, including a number that have not been previously reported; several of these were not related to the tyrosine metabolic pathway. Synopsis Nitisinone therapy has a significant impact on several metabolites beyond the tyrosine metabolic pathway, several of which appear to be related to the redox state of the cell.

Studies in alkaptonuria reveal new roles beyond drug clearance for phase I and II biotransformations in tyrosine metabolism

“…The increases in tyrosine metabolites, excluding HGA, were unexpected in untreated AKU. Increases in metabolites upstream of HPPD previously reported in nitisinonetreated AKU were thought to be a direct consequence of the inhibition of HPPD by nitisinone and the consequential hypertyrosinaemia (Davison et al, 2019a;. For the first time, the present data show that targeted Hgd disruption in Hgd -/mice induces metabolic changes upstream of HGA, despite no increase in tyrosine, HPPA or HPLA.…”

“…Nitisinone reduces plasma and urine HGA concentrations (Phornphutkul et al, 2002;Introne et al, 2011;Olsson et al, 2015;Ranganath et al, 2016;Milan et al, 2017), completely arrests ochronosis in AKU mice (Preston et al, 2014;Keenan et al, 2015) and more recently was shown to slow progression of morbidity in patients attending the UK NAC (2mg nitisinone daily) (Ranganath et al, 2018). We showed previously in serum and urine that nitisinone induced an extended network of metabolic alteration within tyrosine and neighbouring pathways, including tryptophan, purine and TCA cycle (Davison et al, 2019a;Norman et al, 2019). This alteration is a concern in AKU, and particularly in hereditary tyrosinaemia type-1, another inherited disorder of tyrosine metabolism, in which nitisinone treatment is essential from early infancy (McKiernan et al, 2015).…”

“…Previous metabolomic studies in AKU have focused solely on the impact of nitisinone on the metabolome (Davison et al, 2018a(Davison et al, , 2018b(Davison et al, , 2019a(Davison et al, , 2019bNorman et al, 2019).…”

“…However, increased DHMA and VMA indicate increased The finding that p-hydroxyphenylacetic acid and 4-hydroxybenzaldehyde were increased in Hgd -/mice and further increased on nitisinone is interesting. P-Hydroxyphenylacetic acid is generated from gut microbiota and its increase on nitisinone was thought to reflect enhanced microbial metabolism due to reduced oxidative stress upon commencement of treatment (Davison et al, 2019a). 4-Hydroxybenzaldehyde is a naturally occurring metabolite with therapeutic properties in numerous diseases (Kang et al, 2017).…”

“…Application of Abbreviations: AKU, alkaptonuria; HGD, homogentisate 1,2-dioxygenase; HGA, homogentisic acid; LC-QTOF-MS, liquid chromatography quadrupole time-of-flight mass spectrometry; AMRT, accurate mass / retention time; PCA, principal components analysis; RT, retention time; FDR, false-discovery rate; FC, fold change; QC, quality control; CV, coefficient of variation; CSF, cerebrospinal fluid; DHMA, DL-3,4dihydroxymandelic acid; VMA, vanillylmandelic acid; CNS, central nervous system. this technique to AKU serum (Davison et al, 2019a) and urine enabled the discovery of previously unknown metabolite and metabolic pathway alterations following treatment with the HGA-lowering agent nitisinone.…”