Genes & Diseases (2022) 9, 1129e1142

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FULL LENGTH ARTICLE

Metabolomic studies in the inborn error of

metabolism alkaptonuria reveal new
biotransformations in tyrosine metabolism
Brendan P. Norman a,*, Andrew S. Davison b,
Juliette H. Hughes a, Hazel Sutherland a,c, Peter JM. Wilson a,
Neil G. Berry d, Andrew T. Hughes b, Anna M. Milan b,
Jonathan C. Jarvis c, Norman B. Roberts a,
Lakshminarayan R. Ranganath b, George Bou-Gharios a,
James A. Gallagher a

a
Institute of Life Course and Medical Sciences, University of Liverpool, William Henry Duncan Building,
6 West Derby Street, Liverpool, L7 8TX, UK
b
Department of Clinical Biochemistry & Metabolic Medicine, Liverpool Clinical Laboratories, Royal
Liverpool University Hospital, Prescot Street, Liverpool, L7 8XP, UK
c
School of Sport & Exercise Sciences, Liverpool John Moores University, Tom Reilly Building, Byrom
Street, Liverpool, L3 3AF, UK
d
Department of Chemistry, University of Liverpool, Crown Street, Liverpool, L69 7ZD, UK

Received 25 November 2020; received in revised form 13 January 2021; accepted 10 February 2021
Available online 22 February 2021

KEYWORDS Abstract Alkaptonuria (AKU) is an inherited disorder of tyrosine metabolism caused by lack
Alkaptonuria; of active enzyme homogentisate 1,2-dioxygenase (HGD). The primary consequence of HGD
Biotransformation; deficiency is increased circulating homogentisic acid (HGA), the main agent in the pathology
Metabolism; of AKU disease. Here we report the first metabolomic analysis of AKU homozygous Hgd
Metabolomics; knockout (Hgd
/
) mice to model the wider metabolic effects of Hgd deletion and the impli-
Mice cation for AKU in humans. Untargeted metabolic profiling was performed on urine from Hgd
/

AKU (n Z 15) and Hgd þ/
non-AKU control (n Z 14) mice by liquid chromatography high-
resolution time-of-flight mass spectrometry (Experiment 1). The metabolites showing alter-
ation in Hgd
/
were further investigated in AKU mice (n Z 18) and patients from the UK

Abbreviations: AKU, alkaptonuria; HGD, homogentisate 1,2-dioxygenase; HGA, homogentisic acid; LC-QTOF-MS, liquid chromatography
quadrupole time-of-flight mass spectrometry; MS/MS, tandem mass spectrometry; AMRT, accurate mass/retention time; PCA, principal
component analysis; RT, retention time; FDR, false-discovery rate; FC, fold change; QC, quality control; CV, coefficient of variation; MSC,
Molecular Structure Correlator; HPPD, hydroxyphenylpyruvic acid dioxygenase.
* Corresponding author.
E-mail address: bnorman@liv.ac.uk (B.P. Norman).
Peer review under responsibility of Chongqing Medical University.

https://doi.org/10.1016/j.gendis.2021.02.007
2352-3042/Copyright ª 2021, Chongqing Medical University. Production and hosting by Elsevier B.V. This is an open access article under the
CC BY license (http://creativecommons.org/licenses/by/4.0/).
1130 B.P. Norman et al.

National AKU Centre (n Z 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 Z 4) and Hgdþ/
(n Z 4) mice (Experiment 3) to confirm direct
association with HGA. Hgd
/
mice showed the expected increase in HGA, together with un-
expected alterations in tyrosine, purine and TCA-cycle pathways. Metabolites with the great-
est 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 13 C-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 spe-
cific metabolic conditions can elucidate hitherto undiscovered pathways in biochemistry and
metabolism.
Copyright ª 2021, Chongqing Medical University. Production and hosting by Elsevier B.V. This is
an open access article under the CC BY license (http://creativecommons.org/licenses/by/
4.0/).

Introduction ochronosis and its inhibition by nitisinone.10,11 It was

important to establish the metabolic profile of this model,
Alkaptonuria (AKU) is a rare disorder of tyrosine metabolism so we first compared the urinary profiles of AKU homozy-
caused by congenital lack of activity of the enzyme gous Hgd knockout mice (Hgd
/
) with non-AKU heterozy-
homogentisate 1,2-dioxygenase HGD (E.C.1.12.11.5).1 The gous knockout (Hgdþ/
) control mice (Experiment 1).
biochemical consequence of HGD deficiency is increased Secondly, we looked to confirm any AKU-related metabolite
homogentisic acid (HGA) in the circulation, the pathogno- differences by assessing whether the direction of alteration
monic sign of the disease and central to its pathophysio- was reversed while on the HGA-lowering drug nitisinone in
logical features.2 HGA has a high affinity for collagenous Hgd
/
mice or patients (Experiment 2). Thirdly, a meta-
tissues, where its deposition produces striking pigmenta- bolic flux experiment was carried out (Experiment 3), in
tion,3 a process called ochronosis. Cartilage of load-bearing which mice were injected with stable isotope 13C-labelled
joints is particularly susceptible to ochronosis. Presence of HGA to ascertain whether metabolites increased in Hgd
/

HGA-derived pigment in these joints alters the physico- were directly derived from HGA.
chemical properties of cartilage that support normal The overall aim of these experiments (Fig. 1) was to
transmission of load and results in an inevitable and severe investigate in a controlled study the metabolic markers of
early-onset osteoarthropathy.4,5 disease in AKU and if metabolite alterations could elucidate
Metabolomics has emerged as an invaluable approach for possible novel disease mechanisms and thereby improve
studying AKU. In our laboratory we have developed a tar- current strategies for monitoring the disease process and
geted approach with specific mass spectrometric assays as subsequent treatment response in patients with AKU.
an aid for diagnosis and monitoring of AKU.6,7 These assays
offer precise quantification of tyrosine pathway metabo- Materials and methods
lites including HGA in serum and urine. More recently, a
strategy has been developed for profiling and chemical
Animal housing and husbandry
identification of up to 619 related and more general me-
tabolites by high-resolution accurate mass and retention
All mice were housed in the University of Liverpool’s
time using liquid chromatography quadrupole time-of-flight
Biomedical Services Unit under pathogen-free conditions,
mass spectrometry (LC-QTOF-MS).8 Application of this
in cages of up to five mice, with 12-hr light/dark cycle, and
technique to AKU serum9 and urine8 enabled the discovery
food and water available ad libitum. Mice were drug/test-
of previously unknown metabolite and metabolic pathway
naı̈ve at baseline in each experiment.
alterations following treatment with the HGA-lowering
agent nitisinone. Metabolic profiling therefore has poten-
tial in AKU as both a phenotyping and biomarker discovery Materials
tool. However, to our knowledge, untreated AKU has not
been compared with non-AKU at the metabolome level Deionised water was purified in-house by DIRECT-Q 3UV
before. water purification system (Millipore, Watford, UK). Meth-
The investigations into such possible changes in the anol, acetonitrile, isopropanol (SigmaeAldrich, Poole, UK),
metabolome as a result of AKU will be greatly facilitated by formic acid (Biosolve, Valkenswaard, Netherlands) and
use of an animal model of AKU developed in our laboratory ammonium formate (Fisher Scientific, Schwerte, Germany)
by homozygous knockout of the Hgd gene (Hgd
/
) in mice. were LC/MS grade. 13C6 labelled HGA for metabolic flux
The genetically altered Hgd
/
mouse recapitulates human analysis was purchased from Toronto Research Chemicals
AKU, with elevated plasma and urine HGA, development of (Toronto, Canada).
New biotransformations in tyrosine metabolism from alkaptonuri 1131

Figure 1 Schematic overview of the overall study design, incorporating Experiments 1e3. In Experiment 1, urine was collected
from Hgd
/
and Hgdþ/
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 Hgdþ/
. 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 AKU8
(Experiment 2) and a plasma flux analysis using a 13C6 labelled HGA tracer (Experiment 3).

Urine collection and sample preparation for Hgdþ/
groups a separate representative pool was created
metabolomics (Experiment 1) by pooling 20 mL of each individual urine sample. An overall
pool was also created for each experiment by pooling equal
For metabolomic analysis of the targeted Hgd knockout proportions of the above group pools. Analysis of individual
phenotype,10 urine was collected from 15 Hgd
/
(mean and pooled samples was performed following dilution of 1:9
age  SD 12.8  0.1 weeks) and 14 Hgdþ/
(mean age urine:deionised water as previously described.8
11.6  0.3 weeks) male C57BL/6 mice. Drinking water was
supplied ad libitum and the mouse urine was collected on a
single-collection basis onto plastic wrap, pipetted into Investigating the effect of nitisinone on
sample tubes and stored at
80  C. Increased urinary HGA metabolites showing alteration in Hgd
/
mice
was expected to be the most marked metabolic alteration (Experiment 2)
in Hgd
/
, but we were also interested to study the wider,
potentially more subtle metabolic alteration both within The effect of nitisinone treatment on urinary metabolites
the tyrosine pathway and in non-directly associated path- altered in Hgd
/
mice (Fig. 1, Experiment 1) was studied in
ways. The number of mice studied was therefore consid- the data from a previous profiling experiment described by
ered appropriate to sufficiently power this experiment, Norman et al.8 These data were from urine from 18 BALB/c
given that mouse urine collection is a non-invasive Hgd
/
mice (mean age 27  12 weeks, 9 female, 9 male)
procedure. with Hgd disruption by ENU mutagenesis11 and from 25 pa-
Pooled samples were created in each profiling experi- tients attending the UK National Alkaptonuria Centre (NAC;
ment for quality control (QC) purposes. For Hgd
/
and mean age 51  15 years, 13 female, 12 male). The disease
1132 B.P. Norman et al.

phenotypes of Hgd
/
mice from targeted knockout and ENU according to published guidance13 and following the pro-
mutagenesis models are identical.10 Data were from urine cedure described previously by Norman et al.8
collected at baseline, then on nitisinone at one week in mice
(supplied ad libitum in drinking water at 4 mg/L) and at 24 Data processing and statistical analyses
months in patients (2 mg daily dose). These datasets were
acquired under identical LC-QTOF-MS analytical conditions Mining of metabolite features in raw data was performed
to those employed in the present study. using two parallel approaches (Fig. 1, Experiment 1). A
targeted approach was used to extract signals matching a
Design of in vivo metabolic flux experiment and 466-compound AMRT database previously generated in our
sample collection (Experiment 3) laboratory from IROA Technology MS metabolite library
of standards, accessible via https://doi.org/10.6084/m9.
Eight C57BL/6 mice were studied in the HGA metabolic flux figshare.c.4378235.v2,8 or a compound database
experiment; four Hgd
/
(mean age 56  2.3 weeks, 1 fe- comprising accurate masses of additional compounds with
male, 3 male) and four Hgdþ/
(mean age 58  0 weeks, 4 potential relevance to AKU. A complementary non-targeted
female). A 1.96 mg/mL 13C6 HGA tracer solution was pre- approach was used to extract unknown metabolites.
pared in sterile saline and injected into the tail vein. In-
jection volume was adjusted for each mouse to achieve a Targeted feature extraction
final blood concentration of 1 mmol/L, assuming a total Targeted feature extraction was performed in Profinder
blood volume of 75 mL/kg.12 Venous tail bleed samples (build 08.00, Agilent) using the chemical formulae of
were then taken at 2, 5, 10, 20, 40 and 60 min post- compounds from the AMRT database described above.
injection, keeping sampling volumes within LASA guide- Extraction parameters were accurate mass match window
lines.12 Mice were kept anaesthetised with isoflurane 10 ppm and retention time (RT) window 0.3 min.
throughout the experiment. Blood was collected into Allowed ion species were: Hþ, Naþ, and NHþ 4 in positive

Microvette 300 mL lithium heparin capillary tubes (Sarstedt, polarity, and H
and CHO-2 in negative polarity. Charge state
Nümbrecht, Germany) and centrifuged at 1500g for range was 1e2, and dimers were allowed. ‘Find by formula’
10 min. Plasma supernatant was removed and stored at filters were: score >60 in at least 60% of samples in at least

80  C prior to analysis. Individual plasma samples were one sample group (samples were grouped by Hgd
/
or
analysed following 1:9 plasma:deionised water. Hgdþ/
and pre- or on nitisinone).
Seventy-five additional metabolites of potential interest
in AKU or from wider tyrosine metabolism were appended
LC-QTOF-MS analyses to the database for targeted extraction. Forty-three were
from the following pathway databases available from
Analysis of plasma and urine was performed on an Agilent Pathways to PCDL (build 07.00, Agilent): ‘citrate degrada-
1290 Infinity HPLC coupled to an Agilent 6550 QTOF-MS tion’, ‘noradrenaline and adrenaline degradation’ and the
equipped with a dual AJS electrospray ionization source ‘superpathway of phenylalanine, tyrosine and tryptophan
(Agilent, Cheadle, UK). Reversed-phase LC was performed biosynthesis’ (Appendix 2). Six metabolites were added as
on an Atlantis dC18 column (3  100mm, 3 mm, Waters, they were predicted to show potential alteration in Hgd
/

Manchester, UK) maintained at 60  C. Mobile phase compo- due to association with tyrosine conjugation (acetyl-L-
sition was (A) water and (B) methanol, both with 5 mmol/L tyrosine, and g-glutamyl-tyrosine) based on a previous
ammonium formate and 0.1% formic acid. The elution publication8 or association with ochronotic pigment derived
gradient began at 5% B 0e1 min and increased linearly to from HGA (2,5-dihydroxybenzaldehyde, benzoquinone-
100% B by 12 min, held at 100% B until 14 min, then at 5% B acetic acid, hipposudoric acid and norhipposudoric acid).
for a further 5 min. MS data acquisition was performed in Twenty-six metabolites were from a list of potential
positive and negative ionisation polarity with mass range biotransformation products directly derived from HGA and
50e1700 in 2 GHz mode with acquisition rate at 3 spectra/ compiled using the Biotransformation Mass Defects appli-
second. Sample injection volume was 2 mL, and the auto- cation (Agilent; Appendix 3). This tool provides a list of
sampler compartment was maintained at 4  C. Additional potential metabolic biotransformation products covering
data acquisition parameters are detailed in Appendix 1. both phase I and II metabolism for a given compound based
Data-dependent tandem mass spectrometry (MS/MS) on empirical formula. The data were mined for these
was performed on pooled urine samples, with compound additional metabolites with putative identification by ac-
hits from Experiment 1 as [MþH]þ and [M
H]- accurate curate mass (5 ppm) only.
mass precursor ion targets; no more than six compound
targets per injection. Fixed collision energies of 10, 20 and Non-targeted feature extraction
40 V were applied. Acquisition rates were 6 spectra/second Non-targeted extraction was performed by recursive
in MS and 4 spectra/second in MS/MS. feature extraction in Profinder (build 08.00). Extraction
parameters are detailed in Appendix 4.
Design of LC-QTOF-MS profiling analyses
Isotopologue feature extraction on data from 13C6 HGA

/
þ/

Samples from Experiments 1 (Hgd versus Hgd ) and 3 metabolic flux analysis
(13C6 HGA metabolic flux analysis) were analysed in separate Data from Experiment 3 (Fig. 1) were mined using batch
batches, each comprising negative then positive polarity. isotopologue extraction in Profinder (build 08.00). Here,
The analytical sequence of each profiling batch was designed compounds that showed significant differences between
New biotransformations in tyrosine metabolism from alkaptonuri 1133

Hgd
/
and Hgdþ/
mice (Experiment 1) were investigated involves matching between accurate mass MS/MS fragment
for potential association with the 13C6 HGA tracer by ions obtained from collision-induced dissociation MS with
examining the relative abundances of their Mþ0 to Mþ6 one or more proposed molecular structures based on a
isotopologues. Extraction was performed with accurate systematic bond disconnection approach.14
mass and RT match windows of 5 ppm and 0.3 min
respectively against an AMRT database consisting only of Study approval
these compound targets.
All animal work was carried out in accordance with UK
Data QC and statistical analyses Home Office Guidelines under the Animals (Scientific Pro-
First, initial QC was performed on all datasets in Profinder cedures) Act, 1986 and with institutional approval.
by manual curation of the dataset to remove visually low Metabolomic analyses on patient samples was approved
quality peaks and to correct integration issues across the by the Royal Liverpool and Broadgreen University Hospital
dataset where appropriate. Trust’s Audit Committee (audit no. ACO3836) and was part
Urine profiling datasets were then exported from Pro- of the diagnostic service to patients attending the UK Na-
finder as .csv files and imported into Mass Profiler Profes- tional Alkaptonuria Centre in Liverpool.
sional (build 14.5, Agilent) for additional QC and
subsequent statistical analyses. First, creatinine peak area
from this analysis was used as an external scalar for each Results
mouse sample to account for differences in urine concen-
tration, as described previously.8 Additional QC was per- PCA showed clear separation in principal component 1 be-
formed using data from pooled samples, which were tween the urine profiles of Hgd
/
and Hgdþ/
mice from
interspersed throughout each analytical sequence. Com- targeted (Fig. 2A, B) and non-targeted feature extraction.
pounds were retained if a) observed in 100% of replicate Results from targeted and non-targeted extraction are
injections for at least one sample group pool, and b) peak presented separately in the following sections (number of
area coefficient of variation (CV) remained <25% across compounds obtained in each extraction method summar-
replicate injections for each sample group pool. Statisti- ised in Appendix 5). Data from all animals in each experi-
cally significant compounds were then identified in each ment were included in the analysis.
dataset by t-tests; two sample t-tests for Hgd
/
versus
Hgdþ/
comparisons, and paired t-tests for pre- versus on Targeted feature extraction
nitisinone comparisons. Multiple testing correction was
performed using Benjamini-Hochberg false discovery rate Targeted feature extraction was performed to search for
(FDR) adjustment. Fold changes (FC’s) were log2-trans- metabolites based on AMRT (accurate mass 10 ppm, RT
formed and based on peak area. FC cut-off was not applied 0.3 min) or accurate mass (5 ppm) alone. 27/250 and
for compounds with FDR-adjusted P < 0.05, in order to 15/243 metabolites showed abundance differences (FDR-
retain compounds that showed consistent although rela- adjusted P < 0.05) between Hgd
/
and Hgdþ/
in negative
tively low magnitude abundance differences between and positive polarity, respectively. Table 1 shows the
comparison groups. Principal component analysis (PCA) was altered metabolites ranked by FC. PCA loadings plots
performed on each filtered dataset using four-component (Fig. 2C, D) and volcano plots (Fig. 2E, F) show that the
models. greatest differences between Hgd
/
and Hgdþ/
urine
were in metabolites associated with HGA in negative and
In vivo metabolic flux data analysis positive polarity. HGA and six predicted HGA biotransfor-
The results from isotopologue extraction on plasma 13C6 mation products were markedly elevated in Hgd
/
.
HGA metabolic flux data were reviewed visually in Profinder Interestingly, HGA-sulfate (FC Z 9.3, P < 0.0001) showed a
for clear evidence of an isotope label likely to be derived greater FC increase than HGA (FC Z 7.9, P < 0.0001). Other
from HGA. This data review was performed for compound HGA products increased with FC > 1.5 and P < 0.0001 were
matches individually using the predicted number of 13C HGA-glucuronide, HGA-hydroxylsulfate, HGA-N-acetylcys-
atoms derived from the HGA tracer based on chemical teine and HGA-hydrate. Acetyl-HGA was elevated in Hgd
/

structure. (FC Z 2.3, P < 0.0001), but did not pass QC filtering by CV
<25% across replicate injections of pooled QC samples; a
MS/MS data analysis decrease in signal across the run indicated a stability issue
Raw MS/MS data files were processed in MassHunter Quali- (despite the auto-sampler being maintained at 4  C).
tative Analysis Workflows (Build 08.00), using the targeted Excluding HGA, 26 significantly altered compounds were
MS/MS compound discovery algorithm. For compounds from AMRT-matched with metabolites from the 466-compound
the in-house AMRT database, MS/MS spectral library library developed in-house. The most significantly altered
matching was performed against the METLIN metabolite (P < 0.05 and log2 FC > 1.5) of these were: thymidine-50 -
PCDL accurate mass library (build 07.00), which contains diphospho-alpha-D-glucose, DL-3,4-dihydroxymandelic
over 30,000 compound entries, of which 1804 contain acid, 2-aminophenol, p-hydroxyphenylacetic acid
experimental MS/MS spectral data. For the HGA-derived (increased in Hgd
/
), malic acid, citric acid and inosine 50 -
compounds, for which no known chemical standards or monophosphate (decreased in Hgd
/
).
compound library entries currently exist, structure identi- MS/MS fragmentation data were acquired to confirm the
fications were performed using Agilent Molecular Structure compound identifications of the significantly altered me-
Correlator (version B.07.00, build 31). This approach tabolites. For the HGA-derived metabolites, the proposed
1134 B.P. Norman et al.

Figure 2 Clear differences between the urine metabolomes of Hgd
/
and Hgdþ/
mice. (AeD) PCA on data from targeted
feature extraction, with PCA plots showing separation between Hgd
/
and Hgdþ/
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 Hgdþ/
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.

structures (Fig. 4) were imported into the in-house AMRT hydroxymethyl-HGA (53.9%). Lower MSC scores were ob-
compound library as .mol files for matching of experimental tained for the structures proposed for HGA-N-acetylcys-
MS/MS spectra against in silico fragmentation predictions teine (32.4%) and HGA-hydrate (20.3%). The MSC score
using Molecular Structure Correlator (MSC). MSC match obtained for each of these HGA-derived structures was
scores >85% were obtained for HGA-glucuronide (91.2%), either the only compound structure match or was greater
HGA-sulfate (86.7%) and HGA-hydroxylsulfate (85.9%), than the highest scoring match from the MassHunter METLIN
strongly supporting the chemical structures proposed for metabolite library. For the significantly altered metabolites
these compounds. Intermediate scores were obtained for that were not HGA biotransformation products, 15 of the
the proposed structure for acetyl-HGA (54.3%) and AMRT-matched compounds were confirmed by MS/MS
New biotransformations in tyrosine metabolism from alkaptonuri 1135

spectral library match, with a match threshold of 65% injection (Fig. 3). As indicated in Figure 2 (compounds in
(Table 1 & Appendix 6). bold text), two HGA biotransformation products that were
increased in Hgd
/
urine were observed with clear Mþ6
Non-targeted feature extraction peaks over the time course; HGA-glucuronide and HGA-
sulfate. These data confirm that the compounds are
Non-targeted feature extraction yielded 359 and 213 com- derived from HGA. HGA-sulfate showed a similar time
pounds post-QC in negative and positive polarity respec- course profile to HGA; the native Mþ0 isotopologues were
tively; mass range Z 54e3108 Da and RT absent from Hgdþ/
plasma at all time points, in contrast to
range Z 1.1e11.5 min. Comparison of Hgd
/
and Hgdþ/
the Mþ6 isotopologue whose profile appeared to closely
revealed compounds with clear abundance differences follow that of the HGA Mþ6 peak over the time course in
(P < 0.05, log2 FC > 1.5); 9 in negative polarity (6 increased both Hgd
/
and Hgdþ/
(Fig. 3). For HGA-glucuronide, the
in Hgd
/
, 3 decreased in Hgd
/
) and 2 in positive polarity native Mþ0 isotopologue was also absent from Hgdþ/

(both increased in Hgd
/
). The mass range of these sig- plasma, but the Mþ6 isotopologue was only observed in
nificant compounds was 242e507 Da, and RT range was Hgd
/
(Fig. 3), indicating that glucuronidation of the HGA
1.7e9.6 min (Table 1). Searching of the MassHunter METLIN tracer was evident only for Hgd
/
mice.
metabolite PCDL accurate mass library (build 07.00) yielded
no matches for these compounds based on library MS/MS Discussion
spectral matching or accurate mass (<5 ppm) alone.
The data reported form part of the first recorded
The effect of nitisinone treatment on metabolites metabolome-wide comparison obtained from untreated
altered in Hgd
/
mice AKU versus non-AKU animal models and shows, for the first
time, that the overproduced HGA in AKU undergoes pre-
To investigate the effect of nitisinone treatment on the dominantly phase II metabolic biotransformations. Phase II
metabolites shown to be altered here in Hgd
/
, they were metabolism involves conjugation reactions to form sulfate,
searched in the data from previous mouse and human urine glucuronide, glutathione, mercapturic acid, amino acid,
profiling experiments (Experiment 2; Fig. 1).8 methyl and acetyl conjugates.15,16 The predominant sulfa-
Eight compounds from targeted feature extraction that tion and glucuronidation biotransformations observed for
were altered in Hgd
/
versus Hgdþ/
mice were signifi- HGA are also essential aspects of metabolism of other
cantly altered in the opposite direction in urine from both phenolic acids, bile acids, steroids and numerous other
Hgd
/
mice and patients with AKU on nitisinone (based on endogenous biochemicals. The phase I metabolism re-
peak area pre- versus on nitisinone, Benjamini-Hochberg actions of hydroxylation, oxidation, reduction and hydro-
FDR-adjusted P < 0.05; Table 2); seven decreased, and lysis were relatively minor. Further, our data indicate that
one increased. The seven decreased metabolites were the biochemical consequences of HGD deficiency extend
HGA, the HGA biotransformation products HGA-sulfate, beyond tyrosine metabolism.
HGA-glucuronide, HGA-hydrate and hydroxymethyl-HGA,
and also xanthosine and 3,5-cyclic-AMP. The increased HGA biotransformation products from phases I and
metabolite was 3-methyl-glutaric acid. Interestingly, p- II metabolism
hydroxyphenylacetic acid and 4-hydroxybenzaldehyde
were increased both in Hgd
/
versus Hgdþ/
and also on The clearest differences between the urine metabolomes
nitisinone in Hgd
/
mice and patients. of Hgd
/
versus Hgdþ/
mice were in HGA and seven
Nineteen compounds that were altered in Hgd
/
versus previously unreported HGA-derived biotransformation
Hgdþ/
mice were significantly altered in the opposite di- products, increased in Hgd
/
. The decreased output in
rection on nitisinone in urine from either Hgd
/
mice HGA-sulfate, HGA-glucuronide, HGA-hydrate, acetyl-HGA
(n Z 14; 9 decreased, 5 increased on nitisinone) or patients and hydroxymethyl-HGA for patients on treatment with
(n Z 5; 4 decreased, 1 increased on nitisinone) only (Table nitisinone (Experiment 2) indicates that these HGA phase II
2). These compounds comprised the remaining HGA biotransformations occur in human AKU. The detection of
biotransformation products, which were all decreased on 13
C6-labelled forms of HGA-glucuronide and HGA-sulfate in
nitisinone. On nitisinone, acetyl-HGA was decreased in plasma following 13C6-HGA injection in mice (Experiment 3)
patients only, and HGA-hydroxyl-sulfate and HGA-N-ace- confirm products derived from HGA. The 13C6-HGA-sulfate
tylcysteine were decreased in mice only. was observed both in Hgd
/
and Hgdþ/
mice, following
the profile of the 13C6-HGA across the sampling time course.
Confirmation of HGA biotransformation products by The 13C6-HGA-glucuronide was observed only in Hgd
/
,
13
C6 HGA metabolic flux analysis (Experiment 3) suggesting prior upregulation of glucuronyltransferase ac-
tivity was required to enhance HGA clearance in AKU.
Data from isotopologue extraction were compared between The putative structures of these newly identified
plasma collected from the same mice across the time in- biotransformation compounds, together with the enzyme
tervals available (2e60 min). The Mþ6 isotopologue was of classes for their formation are shown in Figure 4, although
particular interest as mice were injected with 13C6-labelled the exact enzymes are not yet described. The proposed
HGA. A clear Mþ6 peak for HGA was observed over the biotransformation compound structures are the closest
sampling time course in plasma from Hgd
/
and Hgdþ/
known matches, based on correlation between the
mice, although the signal decreased from 10 to 20 min post- observed experimental MS/MS spectra and the accurate
1136 B.P. Norman et al.

Table 1 Summary of urinary metabolites showing altered abundance in Hgd
/
mice.

Direction of alteration and log2 fold change is indicated in Hgd
/L (relative to Hgdþ/L); red and blue shading indicates increased
and decreased abundance in Hgd
/L, respectively. p-values are false discovery rate adjusted. Where compounds were signifi-
cantly different in positive and negative polarity, the result with the lowest fold change is provided. For compounds identified by
accurate mass (AM) and retention time (RT), match criteria were accurate mass (10 ppm) and RT (0.3mins) against a database
generated in-house from metabolite standards. Compound identifications based on accurate mass match alone were with a mass
New biotransformations in tyrosine metabolism from alkaptonuri 1137

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 Hgdþ/
samples taken at intervals of 2, 5, 10, 20, 40 and 60 (when
possible) min after injection. Extracted ion chromatograms (EIC’s) represent the Mþ0 (native compound) and Mþ6 (13C6-labelled
form) isotopologue signals for HGA, HGA-sulfate and HGA-glucuronide. EIC’s show clear Mþ6 peaks for these compounds following
injection (but only from Hgd
/
mice for HGA-glucuronide), confirming that they are derived from the labelled HGA.

mass fragment ions predicted in silico from the 30,000þ and how this impacts on predicting the amount of HGA-
metabolite structures in the Metlin and in-house compound derived pigment produced in vivo.
libraries. In the absence of reference standards for these It is not clear whether the HGA biotransformations
compounds, the MS/MS data confirm the structures pro- described are simply unmasked in AKU due to the markedly
posed for HGA-glucuronide, HGA-sulfate and HGA hydrox- increased HGA, or whether they are actively and exclu-
ylsulfate, with MSC scores >85%. To our knowledge, these sively recruited in AKU for HGA detoxification. Neverthe-
are the first data on HGA metabolism not involving con- less, identification of the specific enzymes that catalyse the
version to maleylacetoacetic acid in the traditional tyrosine biotransformations could inform future AKU therapeutic
degradation pathway (Fig. 5). interventions aimed at enhancing HGA metabolism. Glu-
The markedly increased output of the HGA-glucuronide curonidation, for example, is quantitatively one of the most
(FC Z 5) and HGA-sulfate (FC Z 9.3), as well as HGA important phase II biotransformation reactions, performed
(FC Z 7.9), in AKU Hgd
/
mice has implications for the by 15 UPD-glucuronosyltransferase enzymes for conjugation
regulation of plasma HGA and products. Despite efficient of a large number of exogenous and endogenous compounds
renal excretion of HGA-related compounds in AKU,17 the in humans.24,25 A number of agents, including naturally
circulating HGA remains elevated; mean serum HGA con- occurring dietary compounds, are known to be potent in-
centration is 30 mmol/L in untreated AKU18 and <1.5 mmol/ ducers of UPD-glucuronosyltransferases and other phase II
L in healthy subjects.19,20 The increased plasma HGA per- enzymes.26e28 There are encouraging examples of targeting
sists as the primary toxic agent in AKU; its oxidation pro- specific enzymes of phase II metabolism in other conditions
duces ochronotic pigment, which becomes bound within of endogenous compound accumulation, including UDP-
the extracellular matrix of collagenous tissues. This process glucuronosyltransferase 1A1 in neonatal hyper-
of damage is thought to occur via HGA oxidation to highly bilirubinaemia29 and sulfotransferases in oestrogen-
reactive benzoquinone and free radical intermediates.5 dependent breast cancer.30
These species are capable of inducing oxidative changes to The liver is the primary site of phase I and II metabolism,
proteins and lipids,21,22 are known to self-perpetuate both by tissue mass and activity.31 Extrahepatic metabolism
osteoarthropathy associated with ochronosis5 and account in tissues such as the kidney and intestinal epithelium is
for cases of acute fatal metabolic consequences reported in also significant.32 It is possible that site-specific enzyme
the literature.23 The data reported here show that meta- activity explains the interesting finding that conjugation
bolism of HGA by phase II biotransformations, particularly products from the 13C6-labelled HGA were observed mainly
glucuronidation and sulfation reactions, is a major route in for glucuronidation and sulfation. The other biotransfor-
an attempt to render the increased HGA chemically inert mation products observed in urine were not detected in the
(detoxify), in effect to protect from consequences of plasma tracer experiment, despite their clear concomitant
increased benzoquinone production. The HGA-derived urinary reduction on nitisinone confirming direct associa-
products observed must be included in metabolic profiling tion with HGA. In the metabolic flux experiment, HGA was
in AKU patients to understand the total production of HGA administered intravenously, which is likely to favour

window 5 ppm.aMS/MS compound identification based on matching experimental spectra with in silico fragmentation data, using
Molecular Structure Correlator (MSC) with score threshold >65%; all other MS/MS matches were against spectra from the Mass-
Hunter METLIN metabolite PCDL accurate mass library (build 07.00). bAcetyl-HGA failed quality control filtering (CV >25% across
replicate injections of QC pooled samples; due to suspected compound stability issue over analysis period).
1138 B.P. Norman et al.

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 3.053 in silico fragmentation modelling tools.

formation of products from hepatic metabolism. It would female mice, and no difference in metabolism of tyrosine
be interesting therefore to give HGA by various routes of or HGA.8
delivery and trace its metabolism in various tissues and
biofluids to assess the metabolic activity contributed by
different anatomical compartments. It is also worth noting Associated alteration to tyrosine, purine and TCA
that the glycine conjugate of HGA, a potential HGA cycle metabolism in AKU
biotransformation product, was not detected in Hgd
/
;
glycine conjugation is a known detoxification mechanism of This study showed for the first time that in untreated AKU
a number of other aromatic acids.33,34 there is alteration to tyrosine, purine and TCA cycle me-
The observation of marked elevations in five of the tabolites (Fig. 5). Previous metabolomic studies in AKU have
seven HGA-derived compounds which were initially focused solely on the impact of nitisinone on the metab-
discovered in mice, including the major biotransformation olome.8,9,35e37 Nitisinone reversibly inhibits hydrox-
products from HGA sulfation and glucuronidation, in un- yphenylpyruvic acid dioxygenase (HPPD; E.C. 1.13.11.27),
treated patients with AKU (Experiment 2) supports use of the enzyme that produces HGA, and it is currently the most
the Hgd
/
mouse as an accurate model of human AKU effective treatment for AKU. Nitisinone reduces plasma and
biochemistry. Our preclinical studies of the Hgd
/
mouse urine HGA concentrations,18,19,38e40 completely arrests
have helped us to understand the metabolic and wider ochronosis in AKU mice11,41 and more recently was shown to
pathophysiological features of AKU in humans, the natural decrease ochronosis and improve clinical signs of AKU in
course of the disease and its response to treatment. The patients enrolled in SONIA 2, the 4-year phase 3 interna-
mice studied in Experiment 1 were male only in order to tional randomised controlled trial of nitisinone in AKU
enable a highly controlled and powered examination of the (ClinicalTrials.gov identifier: NCT01916382).42 We showed
metabolic impact of Hgd knockout. The inclusion of the previously in serum and urine that nitisinone induced an
previously published data from equal male and female extended network of metabolic alteration within tyrosine
Hgd
/
mice in Experiment 2 confirms that the metabolic and neighbouring pathways, including tryptophan, purine
alterations observed for AKU mice in Experiment 1 are not and TCA cycle.8,9 This alteration is a concern in AKU, and
restricted to males. Further, in Experiment 3 we observed particularly in hereditary tyrosinaemia type-1, another
both native non-labelled and 13C-labelled HGA-glucuronide inherited disorder of tyrosine metabolism, in which nitisi-
and HGA-sulfate products for male and female Hgd
/
none treatment is essential from early infancy.43
mice. As noted in our published discussion of the dataset The increases in tyrosine metabolites reported here,
featured in Experiment 2, the metabolomic difference be- excluding HGA, were unexpected in untreated AKU. In-
tween urine from male and female mice is largely attrib- creases in metabolites upstream of HPPD previously re-
utable to the markedly increased urinary histamine in ported in nitisinone-treated AKU were thought to be a
New biotransformations in tyrosine metabolism from alkaptonuri 1139

Table 2 The effect of nitisinone treatment in Hgd
/
mice and patients with AKU on the abundance of urinary metabolites
altered in Hgd
/
vs. Hgdþ/
mice.

The compounds that showed differences between Hgd
/L vs Hgdþ/L mice (Table 1) were examined in two additional datasets.
Paired t-tests were employed to compare the abundances at baseline versus 1 week on nitisinone (4 mg/L, in drinking water) for
Hgd
/L mice and 24 months on 2 mg daily nitisinone for patients with AKU. Only compounds with false discovery rate adjusted
P < 0.05 pre-vs on nitisinone in mouse or human are displayed. Direction of alteration and log2 fold change are indicated; red
and blue shading indicates increased and decreased abundance on nitisinone, respectively. Where compounds were significantly
different in positive and negative polarity, the result with the lowest fold change is provided. Note: no fold change indicated for
hydroxymethyl-HGA in humans, as this compound was not detected for any patient on nitisinone.ND: compound not detected at
baseline or on nitisinone.
1140 B.P. Norman et al.

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.

direct consequence of the inhibition of HPPD by nitisinone induced oxidative stress in AKU; urinary xanthosine is
and the consequential hypertyrosinaemia.8,9 For the first markedly increased in other oxidative diseases, including
time, the present data show that targeted Hgd disruption in gout,48 chronic kidney disease and diabetes nephropa-
Hgd
/
mice induces metabolic changes upstream of HGA, thy,49 and it is markedly decreased following adminis-
despite no increase in tyrosine, hydroxyphenylpyruvic acid tration of nephroprotective therapy.
or hydroxyphenyllactic acid. The cause of these unex- Decreases in TCA-related metabolites in Hgd
/
is the
pected changes to tyrosine and peripheral neurotransmitter first indication of perturbed energy metabolism in un-
metabolism in untreated AKU is not clear. At the supra- treated AKU. Decreases in citramalic acid, citric acid and N-
physiological concentrations observed in AKU, HGA could acetyl-L-glutamic acid were reversed on nitisinone in Hgd
/


potentially act on other enzymes of neurotransmitter me- , suggesting for the first time that nitisinone is at least
tabolites and alter their activity. Alternatively, the changes partially restorative. Blood and urine concentrations of TCA
could relate to an unknown feature of the disease. metabolites are generally considered to directly reflect
In nitisinone-treated AKU, the purine metabolite overall TCA cycle activity.50 Inhibited TCA cycle activity
changes previously reported were mainly decreased could be due to overall mitochondrial biogenesis,
concentrations.8 The present data indicate a more decreased expression of genes encoding TCA cycle en-
complex pattern of purine alteration in untreated AKU; zymes, dysregulation of enzyme activity or reduced sub-
increased 3,5-cyclic AMP and xanthosine, and decreased strate availability. The latter explanation seems the most
2,3-cyclic AMP and inosine 50 -monophosphate. Purine likely in AKU, in which HGD deficiency prevents further
catabolism is important in the homeostatic response to metabolism of HGA to the TCA cycle intermediate fumaric
various states of mitochondrial oxidative stress, with acid. Decreased bioavailability of fumaric acid may then
shifts occurring to favour breakdown to xanthine and explain the decreases observed for other TCA cycle me-
uric acid, the final breakdown products of purines.44,45 tabolites. Previous analyses have investigated potential
The increased xanthosine and decreases in the upstream changes to TCA cycle metabolites in AKU, but these were
purine pathway metabolites 2,3-cyclic AMP and inosine limited to studying the effect of nitisinone; serum con-
50 -monophosphate reported here for Hgd
/
mice is centrations of TCA metabolites succinic acid and a-keto-
consistent with a shift in purine catabolism as a pro- glutaric acid were decreased in patients with AKU on
tection mechanism against HGA-induced oxidative stress nitisinone.9 The significance of these decreases is not
in untreated AKU, as xanthine derivatives have anti- known, and further studies should investigate whether the
oxidant properties.46 There is growing evidence that alteration to urinary TCA cycle metabolites reported here
HGA-induced oxidative stress is an important feature of also applies to serum in untreated AKU.
AKU (see Ref. 22 for an extensive review on this sub- Decreased urinary citrate is a well-known risk factor for
ject), and that it exacerbates ochronosis, the central kidney stone formation. It is possible that the decreased
AKU disease process.47 The increased xanthosine citric acid reported here for Hgd
/
mice is associated with
observed here is likely a new metabolic marker of HGA- the increased risk of kidney stones in AKU.51 Hypocitraturia
New biotransformations in tyrosine metabolism from alkaptonuri 1141

is generally defined as citric acid excretion <320 mg Study identifier: MTBLS2525 [https://www.ebi.ac.uk/
(1.67 mmol) per day in adults.52 The present profiling data metabolights/MTBLS2525].
are semi-quantitative; targeted quantitative assays are
required to determine exact urinary citric acid concentra- Appendix A. Supplementary data
tions in AKU patients and the additional therapeutic value
of nitisinone in this regard.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.gendis.2021.02.007.
Conclusions

In conclusion, we have shown that targeted homozygous References

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