IJU Case Reports (2023) 6, 436--439

doi: 10.1002/iju5.12642

Case ReportThree cases of xanthinuria identified by gas chromatography/mass
spectrometry-based urine metabolomics
Tomiko Kuhara,1 Masahiro Tetsuo,2 Morimasa Ohse,1 Toshihiko Shirakawa,3 Yumiko Nakashima,3Koh-ichiro Yoshiura,4 Nagaaki Tanaka5 and Tadashi Taya6
1Japan Clinical Metabolomics Institute, Kahoku, 2Testuo Hospital, Minamishimabara, 3Department of Pediatrics, Nagasaki University
Hospital, 4Department of Human Genetics, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, 5Center for Diabetes,
Endocrinology and Metabolism, Kansai Electric Power Hospital, Osaka, and 6Taya Urology Clinic, Komatsu, Ishikawa, Japan

Abbreviations & Acronyms
AKI = acute kidney injury
AMED = Agency for Medical
Research and Development
AO = aldehyde oxidase
EIAKI = exercise-induced
acute kidney injuries
FEUA = fractional excretion ofurate
GC/MS = gas chromatography/
mass spectrometry
GEPH = gephyrin
HUS = hemolytic uremic
syndrome
LC/MS = liquid
chromatography/mass
spectrometry
MOCOS = molybdenum
cofactor sulfurase
XDH = xanthine
dehydrogenase
Correspondence: Tomiko Kuhara
Ph.D., Japan Clinical Metabolomics
Institute, Hamakita I 13-2, Kahoku,
Ishikawa 929-1174, Japan. Email:jcmi-kuhara@bell.ocn.ne.jp
How to cite this article: Kuhara
T, Tetsuo M, Ohse M et al. Threecases of xanthinuria identiﬁed bygas chromatography/mass
spectrometry-based urine
metabolomics. IJU Case Rep.
2023; 6: 436–439.
This is an open access article under
the terms of the Creative
Commons AttributionNonCommercial-NoDerivs License,
which permits use and distribution
in any medium, provided theoriginal work is properly cited, theuse is non-commercial and nomodiﬁcations or adaptations aremade.
Received 4 April 2023; accepted 6
September 2023.
Online publication 28 September
2023

436

Introduction: Early diagnosis of patients with urolithiasis or hypouricemia owing toinborn errors of hypoxanthine metabolism is important in preventing renal failure ordrug-induced toxicity.
Case presentation: We identified three patients with xanthinuria using gas
chromatography/mass spectrometry-based urine metabolomics: a 72-year-old male withbladder stone, a severe hypouricemic 59-year-old female with type 2 diabetes mellitus,
and an 8-year and 9-month-old female who was first discovered to harbor a mutation in
the xanthine dehydrogenase gene using whole-exome sequencing, but had a normal
molybdenum cofactor sulfurase gene. Hydantoin-5-propionate was detected in the first
and third patients but not in the second, suggesting that the first and second patients
had type I and II xanthinuria, respectively.
Conclusion: Gas chromatography/mass spectrometry-based metabolomics can be used
for undiagnosed patients with xanthinuria, identification of the type of xanthinuria
without allopurinol loading, and the quick functional evaluation of mutations in thexanthinuria-related genes.

Key words: hydantoin-5-propionate, hypouricemia, metabolomics, urolithiasis, xanthinuria.

Keynote message
Three patients with xanthinuria were identiﬁed using GC/MS-based urine metabolomics.
Prior to metabolomics, one patient was discovered to harbor a mutation in the xanthine dehydrogenase gene using whole-exome sequencing, necessitating the evaluation of mutation
effect. Typing of type I or type II xanthinuria will be enabled by evaluating hydantoin-5propionate.

Introduction
Xanthinuria is an inborn error of metabolism in the ﬁnal stage of purine catabolism.1 Xanthinuria type I (MIM 278300) is caused by the deﬁciency of XDH.2 Xanthinuria type II (MIM603592) is caused by the mutation of the MOCOS, resulting in the combined deﬁciency of
XDH and AO.3 Molybdenum cofactor deﬁciency (MIM 252150), often called xanthinuria
type III, results from mutations in MOCS1, MOCS2, and the GEPH and causes triple deﬁciency of XDH, AO, and sulﬁte oxidase, frequently resulting in progressive neurological damage and early childhood death.4
Detecting xanthinuria is important to prevent urolithiasis and drug-induced toxicity. Correct
exclusion of xanthinuria is necessary for early diagnosis of hereditary renal hypouricemia orEIAKIs, which have a higher estimated incidence.5,6
We conducted high-risk screening for 130 inborn errors of metabolism, using GC/MSbased metabolomics.7 Herein, we report three cases of xanthinuria.

© 2023 The Authors. IJU Case Reports published by John Wiley & Sons Australia, Ltd on behalf of Japanese Urological Association.

Xanthinuria cases detected by metabolomics

Presentation of cases
Case 1
A 72-year-old male presented with macroscopic hematuria
and urinary retention. Flexible cystourethroscopy revealed
stenosis at the bulbar urethra. The narrowed lumen wasexpanded, and an indwelling bladder catheter was installed.
One month after ﬁrst presentation, the patient was admitted
to undergo cystostomy because of repeated catheter problems.
Abdominal X-ray and cystoscopic examination revealed nostones, but bladder diverticulum. After 1.5 months, a bladderstone measuring 22.8 9 8.8 mm was visualized using cystoscopy. The stone was removed via an endourologic approach
and was conﬁrmed to be struvite. There were no signs indicating urinary stones, except for painless gross hematuria.
Preoperative renal function tests, including estimated glomerular ﬁltration rate (107.21 mL/min/1.73 m2) were normal.
Spot urine and serum were examined using metabolomics toidentify the etiology of stone formation.

Case 2A 55-year-old female with a 10-year history of type 2 diabetes mellitus had a low urate level (0.04 mg/dL), and theFEUA was 6.1%. She also had sudden sensorineural hearing
loss and dyslipidemia. Notably, her sibling had hypouricemia,
and her parents are cousins. The patient had no history ofurolithiasis or EIAKI. At 59 years of age, her spot urine andserum were examined using metabolomics to identify the etiology of hypouricemia.

Case 3A healthy 1-year-old girl developed frequent bloody stools
2 days after the onset of vomiting and diarrhea. Fluid therapyimproved her dehydration. Five days after onset of symptoms, she became pale, and laboratory examination revealed
hemolytic anemia with a hemoglobin level of 5.0 g/dL andthrombocytopenia of 36 9 103/lL. The serum creatininelevel more than doubled from the basal value of 0.22 mg/dL
to 0.55 mg/dL, indicating AKI. Her serum uric acid level
was only 0.2 mg/dL. The patient was diagnosed with thrombotic microangiopathy, probably HUS, following diarrhea
and bloody stools. No Shiga toxin-producing pathogenic E.
coli was detected in the stool. The patient’s serum complement levels were not decreased, and there was no clear evidence of atypical HUS and systemic lupus erythematosus as
the cause of thrombotic microangiopathy.
Moreover, the levels of a disintegrin-like and metalloproteinase with thrombospondin type I motifs 13 (ADAMTS-13)
had not decreased, ruling out thrombotic thrombocytopenic
purpura. Thorough physical examination and imaging studies
showed no evidence of urinary tract obstruction. After supportive care including ﬂuid replacement and red blood celltransfusions, HUS resolved without plasma therapy ordialysis.
There was no known consanguinity in the family. Even
while developing AKI, the serum urate level was low(0.2 mg/dL); after recovery from AKI, it dropped to 0.1–
0.2 mg/dL, with a 2.1% FEUA. Later, the patient had sensorineural hearing loss in her right ear. At the age of 7 years,
she experienced severe left hemi-abdominal pain with grosshematuria. Ultrasound examination did not show any urinarystones. Hematuria and pain resolved spontaneously over
2 days, strongly suggesting renal stones.
Whole-exome sequencing revealed compound heterozygosity of c.3847C > T (p. Arg1283*) and c.1242G > C (p.
Glu414Asp) in the XDH; xanthinuria type I. c.3847C > T,registered as rs751921838 in the SNP database (https://www.
ncbi.nlm.nih.gov/snp/rs751921838#publications), causes nonsense mutation, a novel mutation of XDH.8 c.1242G > C is a
splice-site mutation not registered in the SNP database.
c.1242G > C was predicted as benign by PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://sift.bii.astar.edu.sg/), and as disease-causing by Mutation Taster(http://www.mutationtaster.org/index.html). These resultsappeared compatible with her hypouricemia with low FEUA
level, but were inconclusive. Her MOCOS status was normal,
and xanthinuria type II was ruled out. Her urine was examined using metabolomics to evaluate the function of thisgenetic variant.
All three patients were identiﬁed to have the typical metabolic proﬁle of xanthinuria (Fig. 1; Table 1). Their data werecompared with those of a sibling with xanthinuria type III9that causes triple deﬁciency of XDH, AO, and sulﬁteoxidase.

Discussion
XDH and AO are drug-metabolizing enzymes, whose absence
causes drug-induced toxicity.10 The two types of xanthinuria
are clinically indistinguishable; however, types I and II canbe differentiated using an allopurinol-loading test. Peretz
et al. subtyped xanthinuria into types I and II (i.e., combined
XDH/AO deﬁciency) by assessing biomarkers speciﬁc to AO,
including hydantoin-5-propionate, using LC/MS-based untargeted metabolomics of urine samples.11 We detected normal
urine levels of hydantoin-5-propionate in Cases 1 and 3 but
not in Case 2 or xanthinuria type III. Therefore, Case 1, but
not Case 2, had xanthinuria type I. Hydantoin-5-propionate
evaluation using GC/MS would facilitate rapid differentiationof type I and type II/type III.
Prior to metabolomics, Case 3 was discovered to harbor amutation in XDH, but not in MOCOS, via whole-exome
sequencing. Metabolomics can be used to assess the biochemical or clinical signiﬁcance of a mutation,12 and thus,
complements whole-exome sequencing. However, failure todetect a pathogenic mutation using conventional genetic analysis does not exclude a disease, particularly where an abnormal metabolic phenotype has been detected.13
Patients with xanthinuria type I and II can be asymptomatic or symptomatic.14 In a Czech population, nine cases(four asymptomatic) of hereditary xanthinuria were detected,
suggesting that this is a common disorder of purine
metabolism.15 Over 50% of the reported cases were symptomatic; therefore, early diagnosis may prevent disease progression, severe complications, and end-stage renal disease.

© 2023 The Authors. IJU Case Reports published by John Wiley & Sons Australia, Ltd on behalf of Japanese Urological Association.

437

T KUHARA ET AL.

(a)

(b)

(c)

Fig. 1 Mass chromatograms of the trimethylsilyl
derivatives of hypoxanthine, xanthine, urate, andcreatinine from urine and serum. Sample
preparation and GC/MS measurement were thesame as those described previously.7 Metabolites
in the urine were evaluated as creatinine-based.7Metabolites in the serum were evaluated using 2hydroxyundecanoate, 25 lmol of which wasadded per 0.1 mL of serum. (a) Urine from Case
1, (b) urine from an age-matched control, (c)serum from Case 1, and (d) serum from an agematched control.

(d)

Table 1 Z-scores of the biomarkers, hypoxanthine, xanthine, urate, and hydantoin-5-propionate†
Case 1Case 2

Target

Urine

Serum

Urine

Serum

Case 3Urine

MOCS1‡Urine

MOCS1‡
Urine

Hypoxanthine
Xanthine
UrateΣ§Hydantoin-5-propionate¶

4.0
8.1
5.6
12.1
0.9

7.8
3.7
0.9
11.5
—4.3
10.0
12.0
14.3
3.7

2.0
5.0
3.5
7.0—

5.0
6.0
8.0
11.0
1.3

5.9
3.5
5.5
9.4
< 44.34.5
14.5
8.8< 4Urine was more sensitive than serum as an indicator of urate production inability or hypouricemia. †Z-score was calculated after log10-transformation, asdescribed.12 In the metabolomics studies performed, the quantitative values of biomarkers are not obtained. Instead, creatinine-based relative values areVmean) / SD, Vp: measured value in a patient;obtained. Z-score (n) indicates the degree of deviation of a patient’s biomarkers from control values. n = (VpVmean: mean of measured values in control group. Vp and each value in control groups are all log10 transformed. For example, in case 2, Z-score (4.3) of hypoxanthine was derived from Vp, log10(Vp), Vmean, SD, log10(Vp) Vmean, the values of which were 2.11, 0.324, 0.557, 0.204, and 0.881, respectively. ‡MOCS1
denotes the molybdenum cofactor synthesis 1 gene responsible for xanthinuria type III. Data are from siblings diagnosed with mutations in MOCS1.9 §Sum ofZ-scores of hypoxanthine and xanthine. ¶The AO-specific biomarker. SD, standard deviation.

438© 2023 The Authors. IJU Case Reports published by John Wiley & Sons Australia, Ltd on behalf of Japanese Urological Association.

Xanthinuria cases detected by metabolomics

We demonstrated the usefulness of urine metabolomics forpatients with urolithiasis or hypouricemia owing to xanthinuria. Patients who form stones owing to inborn errors of
metabolism may be a minority among all patients with urolithiasis. However, we propose that, for patients with stoneforming conditions whose diagnosis has been delayed formonths, years, or more, and those with hypouricemia, GC/MS-based urine metabolomics can help rapidly identify thosehaving inborn errors of metabolism.

Acknowledgments
Part of this study was supported by Grants-in-Aid(20ek0109301s4003) from the Japan Agency for Medical
Research and Development.

Author contributions
Tomiko Kuhara: Conceptualization; formal analysis; methodology; resources; validation; writing – original draft; writing –review and editing. Masahiro Tetsuo: Conceptualization; investigation; resources; writing – original draft; writing – review
and editing. Morimasa Ohse: Formal analysis; investigation;
visualization. Toshihiko Shirakawa: Investigation; resources;
writing – original draft. Yumiko Nakashima: Investigation;
resources; writing – original draft. Koh-ichiro Yoshiura: Investigation; resources; writing – original draft. Nagaaki Tanaka:Investigation; resources; writing – original draft. Tadashi Taya:Conceptualization; investigation; resources; supervision.

Conflict of interest
The authors declare no conﬂict of interest.

Approval of the research protocol by
an Institutional Reviewer Board
All procedures in this study that involved human participantswere performed in accordance with the ethical standards ofthe Institutional Review Board and the 1964 Helsinki declaration and its later amendments or comparable ethicalstandards.

Informed consent
Written informed consent was obtained from all individual
participants included in the study.

Registry and the Registration No. ofthe study/trial
Not applicable.

Funding information
None.

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