Molecular Genetics and Metabolism 82 (2004) 220223

www.elsevier.com/locate/ymgme

Guanidinoacetate and creatine/creatinine levels in controls

and patients with urea cycle defects
Angela Arias,a,b Judit Garcia-Villoria,a and Antonia Ribesa,
a

Instituto de Bioqumica Clnica, Corporaci Sanitria Clinic, Barcelona, Spain

b
Centro de Desarrollo Infantil, MECD, Mrida, Venezuela

Received 9 February 2004; received in revised form 16 April 2004; accepted 16 April 2004

Abstract
We established an analytical methodology for guanidinoacetate and creatine determination by gas chromatographymass spectrometry with the use of stable isotopes as internal standards. The method showed good precision and high sensitivity, and it requires
minimal sample handling. We determined the reference values in urine and plasma. In urine both guanidinoacetate concentration
and creatine/creatinine ratio decrease as age increases, but no signiWcant diVerences were found in plasma. In addition, 15 patients
with urea cycle defects were analysed and showed low guanidinoacetate concentrations when compared with age-matched controls.
We concluded that guanidinoacetate concentration is a parameter to be considered in the follow-up of patients with urea cycle
defects, and arginine should be supplemented in suYcient amounts, as the brain seems to be impermeable to creatine inXux, but not
to its precursor, arginine, which is needed for creatine, protein, and NO synthesis.
2004 Elsevier Inc. All rights reserved.
Keywords: GAMT; AGAT; Guanidinoacetate; GC/MS; Urea cycle defects

Introduction
Disorders of creatine pathway can be categorised into
disorders of creatine synthesis, including arginine:glycine amidinotransferase (AGAT, OMIM 602360) and
guanidinoacetate methyltransferase (GAMT, OMIM
601240) deWciencies, which are inherited as autosomal
recessive traits, and an X-linked inherited defect of cellular transport caused by derangement of creatine transporter protein (CrT1, OMIM 300036). A common
feature in all these disorders is the complete lack of creatine/creatine phosphate in the brain, measured by in vivo
magnetic resonance spectroscopy (MRS). Mental retardation and speech delay are the common clinical denominators of all creatine deWciency syndromes. Patients
with AGAT and CrT1 deWciency may additionally have
epileptic seizures with satisfactory response to common
antiepileptic drugs, while patients with GAMT deWciency

Corresponding author. Fax: +34-93-227-5668.

E-mail address: aribes@clinic.ub.es (A. Ribes).

1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymgme.2004.04.009

exhibit a more complex clinical phenotype with dystonic

hyperkinetic movement disorder and epilepsy that is
unresponsive to treatment with antiepileptic drugs [13].
Biochemically, GAMT deWciency is characterised by the
speciWc accumulation of guanidinoacetate (GAA) in biological Xuids, while this compound is extremely low in
AGAT deWciency [4]. In contrast, GAA concentration is
normal in CrT1 deWciency, but the high creatine/creatinine ratio can be used as the Wrst biochemical diagnostic
marker for this disease [5].
The diagnosis in aVected children is commonly made
by measuring GAA and creatine concentrations in
plasma and urine using high performance liquid chromatography (HPLC) [6], tandem mass spectrometry
(TMS) [7], gas chromatographymass spectrometry
(GCMS) [8] or stable isotope dilution GCMS [9]. Further conWrmation of the diagnosis can be made by mutation analysis or by measuring the enzyme activity in
Wbroblasts, lymphocytes or cultured amniocytes [1012].
The purpose of this work was to establish an analytical methodology by GCMS for the evaluation of creatine metabolism, to obtain the control values, and to

A. Arias et al. / Molecular Genetics and Metabolism 82 (2004) 220223

compare these with values for patients with urea cycle

defects.

Materials and methods

Control subjects
Urine samples from 75 healthy children were
obtained thanks to the collaboration of Urgell School in
Barcelona, Spain. The age of children ranged from 2 to
12 years. For each child informed consent from the
parents was obtained. Twenty samples from newborns
were collected among children that were sent to our laboratory to be screened for inborn errors of metabolism
and who retrospectively proved to be normal; 59 adult
urine samples were obtained from healthy volunteers
working in our hospital. Plasma samples from 17 healthy
children and adults (age range: from 1 to 29 years) were
also studied.
Patients with urea cycle defects
We studied 15 patients with urea cycle defects, diagnosed in our laboratory. We included eight patients with
ornithine transcarbamylase deWciency (OTC, OMIM
311250), three patients with argininosuccinicaciduria
(ASL, OMIM 207900), three patients with citrullinemia
(ASS, OMIM 215700) and one patient with hyperornithinemia, hyperammonenia, and homocitrullinuria
(HHH, OMIM 238970). When possible, one sample preand another post-treatment were studied.

221

for 10 min at 3000 rpm, and 400
L of the supernatant

was transferred to a clean vial and blown to dryness with
N2. Finally, the samples were derivatized with 150
L of
BSTFA for 30 min at 60 C. The standard curves for creatine and GAA over a range of 10385 and 586 nmol,
respectively, were prepared using Wve diVerent concentrations in duplicate. All standard solutions were dissolved in water.
The intra-assay precision was evaluated by performing 20 analyses of the same sample on the same day. For
the establishment of the inter-assay variability, one sample was processed in 10 independent preparations on 10
diVerent days. Recovery was evaluated by adding known
amounts of GAA and creatine (42 and 38 nmol, respectively) to a urine sample. All the analyses were performed in triplicate.
Gas chromatographymass spectrometry GC/MS
Chromatographic separation was performed on a
capillary column (30 m 0.25 mm ID, Wlm thickness
0.25
m Supelco SPB-1) with the use of a HewlettPackard GC/MS 5989 A (Palo Alto, CA). The initial oven
temperature was maintained at 140 C, 1.9 C/min, followed by a ramp of 20 C/min to 250 C. The mass
spectrometer was operated under electronic impact ionization in the single-ion monitoring mode. The ions measured were m/z 225 and m/z 226 for GAA and
[13C2]guanidino acetic acid, and m/z 258 and 261 for creatine and d3-creatine, respectively. The total run time
was 16.03 min.
Statistical analysis

Reagents
1,1,1,5,5,5-HexaXuoro 2,4-pentane (98%), creatine
hydrate, guanidino acetic acid, and bis(trimethylsilil) triXuoroacetamide (BSTFA) were purchased from Sigma
Aldrich, Madrid (Spain). N-methyl-d3-creatine (99%)
was from CDN Isotopes, Paris (France), and [13C2]guanidino acetic acid was provided by HJ Ten Brink, Free
University of Amsterdam (The Netherlands). All other
solvents and chemicals were of analytical grade and were
obtained from a variety of sources.

The data are expressed as median and interval. After

statistically analysing the GAA and creatine/creatinine
ratio according to age, we established three age groups
in urine for which these parameters were most signiWcantly diVerent: 2 days-6 years, 712 years, and adults.
The number of subjects in each age group is shown in
Table 1. No signiWcant diVerences among age groups
were found in plasma.

Results and discussion

Analytical method
We used the method of Hunneman and Hanefeld [8]
and Struys et al. [9] with slight modiWcations. BrieXy,
100
L of urine or plasma was mixed with 50
L of saturated aqueous sodium bicarbonate, 50
L of hexaXuoroacetylacetamide, and 600
L toluene. One hundred
microlitre of labelled internal standard solution comprising 152.7 nmol d3-creatine and 42.7 nmol [13C2]guanidinoacetate was added. The mixture was heated to 80 C for
2 h. The toluene phase was separated by centrifugation

Several methods for the diagnosis of creatine deWciencies, including methods based on HPLC [6], TMS
[7], and GC/MS [8,9], have been reported. The latter
was the method of choice in our laboratory. Linear correlation between peak areas and GAA and creatine
concentrations were found. The correlation coeYcients
for GAA and creatine were 0.998 and 0.997, respectively. The detection limit in a 100
L sample volume
for GAA and creatine was 0.1
M (or 1 pmol) with a
signal-to-noise ratio of 2. Intra-assay and inter-assay

222

A. Arias et al. / Molecular Genetics and Metabolism 82 (2004) 220223

Table 1
Urine concentration of GAA and creatine/creatinine ratio in controls and patients with urea cycle defects before and after citrulline or arginine substitution
Urine

Control values

Age range
Number of individuals (n)
GAA mmol/mol creatinine
Creatine/creatinine ratio

2 days6 years
68
73 (21124)
0.44 (0.021.9)

ASL, ASS, OTC, and HHH

712 years
27
56 (1890)
0.13 (0.021.2)

Adults
59
40 (1184)
0.03 (0.020.4)

Before

After

2 days2 years
10
2 (0.59)
0.2 (0.021.2)

4 months15 years
4
76 (5183)
0.5 (0.30.7)

OTCa

1 month4 years
4
38 (2265)
0.3 (0.050.4)

Values are expressed as median and range.

Nearly asymptomatic female patients heterozygous for OTC deWciency.

for GAA and creatine were 4.7 and 6.5% and 4.5 and
6.9%, respectively. The mean recovery for GAA and
creatine was 100.9 and 102%, respectively. Therefore,
this method shows good precision and sensitivity,
requires only minimal sample handling and is able to
detect values below and above those found in healthy
individuals (Tables 1 and 2). We improved the method
of Hunneman and Hanefeld [8] basically by reducing
the hours of derivatization and by analysing creatine
and GAA in the same preparation step, as well as by
using stable isotope labelled internal standards. Struys
et al. [9] were the Wrst to use [13C2]guanidino acetic acid
and d3-creatine as internal standards, but their method,
although more sensitive than ours, is not suitable for
most biochemical genetic laboratories using simple
GCMS because negative chemical ionisation is
required. On the other hand, while Struys et al. [9] use a
very polar GC column, we use a common non-polar
column, which is an advantage for laboratories doing
routine organic acid analysis, in that it is not necessary
to change the column when GAA and creatine determination is required.
In contrast to Carducci et al. [6], we found a negative
correlation between age and GAA concentration and
creatine/creatinine ratio in urine, as both parameters
decrease as age increases (Table 1), but no signiWcant
diVerences were found in plasma, probably due to the
low number of controls compared with urine (Table 2).
The validity of this method has been demonstrated by
detecting a patient aVected with GAMT deWciency with
a concentration of 228 mmol GAA/mol creatinine (CV
for his age, median: 56, interval: 1890), and two
patients with CrT1 defect with a creatine/creatinine
ratio of 3.4 and 2.9, respectively (CV for his age,

median: 0.13, interval: 0.021.2). Both diagnoses were

conWrmed by enzymatic or mutational studies in
another laboratory.
Arginine plays key roles in CNS not only as a substrate for protein synthesis or precursor of NO, but also
as a substrate for creatine synthesis by providing guanidino groups [13,14]. Furthermore, it has been shown
that exposure of rat brain cell aggregates to ammonia
leads to a decrease of intracellular creatine concentration and that creatine has a protective action on axonal
development [15]. For this reason we decided to investigate creatine metabolism in patients with urea cycle
defects, as one consequence of these defects (except for
arginase deWciency) is a decreased level of arginine.
Indeed, we studied patients with OTC, ASL, and ASS
deWciencies, and a patient with HHH syndrome, and
found that before treatment GAA concentration in
urine was signiWcantly lower compared to age matched
controls (Table 1), while after arginine or citrulline substitution GAA normalised. Exceptions were some
female patients with OTC deWciency, probably due to a
less severe disease, as expected from the skewed X-inactivation. In plasma, arginine and GAA were low in most
cases, but both parameters normalised following arginine or citrulline supplementation (Table 2), thereby
providing evidence of the importance of arginine for
creatine biosynthesis. Plasma from a patient with HHH
who was previously published [16] was not available, but
it seems that in this case the reason for low GAA was
the high ornithine concentration that could inhibit
AGAT activity [17].
These results suggest that the GAA concentration is a
parameter to consider in the follow-up of patients with
urea cycle defects, and arginine should be supplemented

Table 2
Plasma concentration of arginine, GAA, and creatine in controls and patients with urea cycle defects, before and after citrulline or arginine substitution
Plasma

Arginine (
mol/L)
GAA (
mol/L)
Creatine (
mol/L)

Control values n: 17

58 (29133)
1.7 (0.72.5)
76 (45228)

OTCa n: 1

ASL, ASS, and OTC

Before n: 9

After n: 12

50 (12140)
0.77 (0.030.87)
59 (28100)

100 (40184)
1.30 (1.022.19)
44 (28104)

Values are expressed as median and range.

a
Nearly asymptomatic female patient heterozygous for OTC deWciency.

126
1.49
77

A. Arias et al. / Molecular Genetics and Metabolism 82 (2004) 220223

in suYcient amounts, as the brain seems to be impermeable to creatine inXux, but not to its precursor, arginine.

Acknowledgments
We thank C. Llords for her invaluable help in sample analyses. We also thank Dr. M. Rods for amino acid
analyses. The Wnancial support of FIS (Grant # 2003REDG054B-O) is acknowledged.

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