www.elsevier.com/locate/ymgme
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
1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymgme.2004.04.009
221
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.
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
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)
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)
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,
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
After n: 12
50 (12140)
0.77 (0.030.87)
59 (28100)
100 (40184)
1.30 (1.022.19)
44 (28104)
126
1.49
77
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.
References
[1] M.C. Bianchi, M. Tosetti, F. Fornai, M.G. Alessandri, P. Cipriani,
G. De Vito, R. Canapicchi, Reversible brain creatine deWciency in
two sisters with normal blood creatine level, Ann. Neurol. 47
(2000) 511513.
[2] G.S. Salomons, S.J.M. van Dooren, N.M. Verhoeven, K.M. Cecil,
W.S. Ball, T.J. Degrauw, C. Jakobs, X-linked creatine-transporter
gene (SLC6A8) defect: a new creatine-deWciency syndrome, Am. J.
Hum. Genet. 68 (2001) 14971500.
[3] C. Stromberger, O.A. Bodamer, S. Stockler-Ipsiroglu, Clinical
characteristics and diagnostic clues in inborn errors of creatine
metabolism, J. Inherit. Metab. Dis. 26 (2003) 299308.
[4] C.B. Item, S. Stockler-Ipsiroglu, C. Stromberger, A. Muhl, M.G.
Alessandri, M.C. Bianchi, M. Tosetti, F. Fornai, G. Cioni, Arginine:glycine amidinotransferase deWciency: the third inborn error
of creatine metabolism in humans, Am. J. Hum. Genet. 69 (2001)
11271133.
[5] K.A. Hahn, G.S. Salomons, D. Tackels-Horne, T.C. Wood, H.A.
Taylor, R.J. Schroer, H.A. Lubs, C. Jakobs, R.L. Olson, K.R.
Holden, R.E. Stevenson, C.E. Schwartz, X-linked mental retardation with seizures and carrier manifestations is caused by a mutation in the creatine-transporter gene (SLC6A8) located in Xq28,
Am. J. Hum. Genet. 70 (2002) 13491356.
[6] C. Carducci, M. Birarelli, V. Leuzzi, C. Carducci, R. Battini, G.
Cioni, I. Antonozzi, Guanidinoacetate and creatine plus creatinine
assessment in physiologic Xuids: an eVective diagnostic tool for
the biochemical diagnosis of arginine:glycine amidinotransferase
and guanidinoacetate methyltransferase deWciencies, Clin. Chem.
48 (2002) 17721778.
223