Molecular Genetics and Metabolism 82 (2004) 220–223

www.elsevier.com/locate/ymgme

Guanidinoacetate and creatine/creatinine levels in controlsand patients with urea cycle defects

Angela Arias,a,b Judit Garcia-Villoria,a and Antonia Ribesa,¤

a Instituto de Bioquímica Clínica, Corporació Sanitària Clinic, Barcelona, Spainb Centro de Desarrollo Infantil, MECD, Mérida, 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 chromatography–mass spec-trometry with the use of stable isotopes as internal standards. The method showed good precision and high sensitivity, and it requiresminimal sample handling. We determined the reference values in urine and plasma. In urine both guanidinoacetate concentrationand creatine/creatinine ratio decrease as age increases, but no signiWcant diVerences were found in plasma. In addition, 15 patientswith 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 cycledefects, and arginine should be supplemented in suYcient amounts, as the brain seems to be impermeable to creatine inXux, but notto 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 intodisorders of creatine synthesis, including arginine:gly-cine amidinotransferase (AGAT, OMIM 602360) andguanidinoacetate methyltransferase (GAMT, OMIM601240) deWciencies, which are inherited as autosomalrecessive traits, and an X-linked inherited defect of cellu-lar transport caused by derangement of creatine trans-porter protein (CrT1, OMIM 300036). A commonfeature in all these disorders is the complete lack of crea-tine/creatine phosphate in the brain, measured by in vivomagnetic resonance spectroscopy (MRS). Mental retar-dation and speech delay are the common clinical denom-inators of all creatine deWciency syndromes. Patientswith AGAT and CrT1 deWciency may additionally haveepileptic seizures with satisfactory response to commonantiepileptic 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 dystonichyperkinetic movement disorder and epilepsy that isunresponsive to treatment with antiepileptic drugs [1–3].Biochemically, GAMT deWciency is characterised by thespeciWc accumulation of guanidinoacetate (GAA) in bio-logical Xuids, while this compound is extremely low inAGAT deWciency [4]. In contrast, GAA concentration isnormal in CrT1 deWciency, but the high creatine/creati-nine ratio can be used as the Wrst biochemical diagnosticmarker for this disease [5].

The diagnosis in aVected children is commonly madeby measuring GAA and creatine concentrations inplasma and urine using high performance liquid chro-matography (HPLC) [6], tandem mass spectrometry(TMS) [7], gas chromatography–mass spectrometry(GC–MS) [8] or stable isotope dilution GC–MS [9]. Fur-ther conWrmation of the diagnosis can be made by muta-tion analysis or by measuring the enzyme activity inWbroblasts, lymphocytes or cultured amniocytes [10–12].

The purpose of this work was to establish an analyti-cal methodology by GC–MS for the evaluation of crea-tine metabolism, to obtain the control values, and to

A. Arias et al. / Molecular Genetics and Metabolism 82 (2004) 220–223 221

compare these with values for patients with urea cycledefects.

Materials and methods

Control subjects

Urine samples from 75 healthy children wereobtained thanks to the collaboration of Urgell School inBarcelona, Spain. The age of children ranged from 2 to12 years. For each child informed consent from theparents was obtained. Twenty samples from newbornswere collected among children that were sent to our lab-oratory to be screened for inborn errors of metabolismand who retrospectively proved to be normal; 59 adulturine samples were obtained from healthy volunteersworking in our hospital. Plasma samples from 17 healthychildren and adults (age range: from 1 to 29 years) werealso studied.

Patients with urea cycle defects

We studied 15 patients with urea cycle defects, diag-nosed in our laboratory. We included eight patients withornithine transcarbamylase deWciency (OTC, OMIM311250), three patients with argininosuccinicaciduria(ASL, OMIM 207900), three patients with citrullinemia(ASS, OMIM 215700) and one patient with hyperorni-thinemia, hyperammonenia, and homocitrullinuria(HHH, OMIM 238970). When possible, one sample pre-and another post-treatment were studied.

Reagents

1,1,1,5,5,5-HexaXuoro 2,4-pentane (98%), creatinehydrate, guanidino acetic acid, and bis(trimethylsilil) tri-Xuoroacetamide (BSTFA) were purchased from Sigma–Aldrich, Madrid (Spain). N-methyl-d3-creatine (99%)was from CDN Isotopes, Paris (France), and [13C2]gua-nidino acetic acid was provided by HJ Ten Brink, FreeUniversity of Amsterdam (The Netherlands). All othersolvents and chemicals were of analytical grade and wereobtained from a variety of sources.

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 satu-rated aqueous sodium bicarbonate, 50 �L of hexaXuoro-acetylacetamide, and 600 �L toluene. One hundredmicrolitre of labelled internal standard solution compris-ing 152.7 nmol d3-creatine and 42.7 nmol [13C2]guanidino-acetate was added. The mixture was heated to 80 °C for2 h. The toluene phase was separated by centrifugation

for 10 min at 3000 rpm, and 400 �L of the supernatantwas transferred to a clean vial and blown to dryness withN2. Finally, the samples were derivatized with 150 �L ofBSTFA for 30 min at 60 °C. The standard curves for cre-atine and GAA over a range of 10–385 and 5–86 nmol,respectively, were prepared using Wve diVerent concen-trations in duplicate. All standard solutions were dis-solved in water.

The intra-assay precision was evaluated by perform-ing 20 analyses of the same sample on the same day. Forthe establishment of the inter-assay variability, one sam-ple was processed in 10 independent preparations on 10diVerent days. Recovery was evaluated by adding knownamounts of GAA and creatine (42 and 38 nmol, respec-tively) to a urine sample. All the analyses were per-formed in triplicate.

Gas chromatography–mass spectrometry GC/MS

Chromatographic separation was performed on acapillary column (30 m £ 0.25 mm ID, Wlm thickness0.25 �m Supelco SPB-1) with the use of a Hewlett–Pack-ard GC/MS 5989 A (Palo Alto, CA). The initial oventemperature was maintained at 140 °C, 1.9 °C/min, fol-lowed by a ramp of 20 °C/min to 250 °C. The massspectrometer was operated under electronic impact ioni-zation in the single-ion monitoring mode. The ions mea-sured were m/z 225 and m/z 226 for GAA and[13C2]guanidino acetic acid, and m/z 258 and 261 for cre-atine and d3-creatine, respectively. The total run timewas 16.03 min.

Statistical analysis

The data are expressed as median and interval. Afterstatistically analysing the GAA and creatine/creatinineratio according to age, we established three age groupsin urine for which these parameters were most signiW-cantly diVerent: 2 days-6 years, 7–12 years, and adults.The number of subjects in each age group is shown inTable 1. No signiWcant diVerences among age groupswere found in plasma.

Results and discussion

Several methods for the diagnosis of creatine deW-ciencies, including methods based on HPLC [6], TMS[7], and GC/MS [8,9], have been reported. The latterwas the method of choice in our laboratory. Linear cor-relation between peak areas and GAA and creatineconcentrations were found. The correlation coeYcientsfor GAA and creatine were 0.998 and 0.997, respec-tively. The detection limit in a 100 �L sample volumefor GAA and creatine was 0.1 �M (or 1 pmol) with asignal-to-noise ratio of 2. Intra-assay and inter-assay

222 A. Arias et al. / Molecular Genetics and Metabolism 82 (2004) 220–223

for GAA and creatine were 4.7 and 6.5% and 4.5 and6.9%, respectively. The mean recovery for GAA andcreatine was 100.9 and 102%, respectively. Therefore,this method shows good precision and sensitivity,requires only minimal sample handling and is able todetect values below and above those found in healthyindividuals (Tables 1 and 2). We improved the methodof Hunneman and Hanefeld [8] basically by reducingthe hours of derivatization and by analysing creatineand GAA in the same preparation step, as well as byusing stable isotope labelled internal standards. Struyset al. [9] were the Wrst to use [13C2]guanidino acetic acidand d3-creatine as internal standards, but their method,although more sensitive than ours, is not suitable formost biochemical genetic laboratories using simpleGC–MS because negative chemical ionisation isrequired. On the other hand, while Struys et al. [9] use avery polar GC column, we use a common non-polarcolumn, which is an advantage for laboratories doingroutine organic acid analysis, in that it is not necessaryto change the column when GAA and creatine determi-nation is required.

In contrast to Carducci et al. [6], we found a negativecorrelation between age and GAA concentration andcreatine/creatinine ratio in urine, as both parametersdecrease as age increases (Table 1), but no signiWcantdiVerences were found in plasma, probably due to thelow number of controls compared with urine (Table 2).The validity of this method has been demonstrated bydetecting a patient aVected with GAMT deWciency witha concentration of 228 mmol GAA/mol creatinine (CVfor his age, median: 56, interval: 18–90), and twopatients with CrT1 defect with a creatine/creatinineratio of 3.4 and 2.9, respectively (CV for his age,

median: 0.13, interval: 0.02–1.2). Both diagnoses wereconWrmed by enzymatic or mutational studies inanother laboratory.

Arginine plays key roles in CNS not only as a sub-strate for protein synthesis or precursor of NO, but alsoas a substrate for creatine synthesis by providing guani-dino groups [13,14]. Furthermore, it has been shownthat exposure of rat brain cell aggregates to ammonialeads to a decrease of intracellular creatine concentra-tion and that creatine has a protective action on axonaldevelopment [15]. For this reason we decided to investi-gate creatine metabolism in patients with urea cycledefects, as one consequence of these defects (except forarginase deWciency) is a decreased level of arginine.Indeed, we studied patients with OTC, ASL, and ASSdeWciencies, and a patient with HHH syndrome, andfound that before treatment GAA concentration inurine was signiWcantly lower compared to age matchedcontrols (Table 1), while after arginine or citrulline sub-stitution GAA normalised. Exceptions were somefemale patients with OTC deWciency, probably due to aless severe disease, as expected from the skewed X-inac-tivation. In plasma, arginine and GAA were low in mostcases, but both parameters normalised following argi-nine or citrulline supplementation (Table 2), therebyproviding evidence of the importance of arginine forcreatine biosynthesis. Plasma from a patient with HHHwho was previously published [16] was not available, butit seems that in this case the reason for low GAA wasthe high ornithine concentration that could inhibitAGAT activity [17].

These results suggest that the GAA concentration is aparameter to consider in the follow-up of patients withurea cycle defects, and arginine should be supplemented

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

Values are expressed as median and range.a Nearly asymptomatic female patients heterozygous for OTC deWciency.

Urine Control values ASL, ASS, OTC, and HHH OTCa

Before After

Age range 2 days–6 years 7–12 years Adults 2 days–2 years 4 months–15 years 1 month–4 yearsNumber of individuals (n) 68 27 59 10 4 4GAA mmol/mol creatinine 73 (21–124) 56 (18–90) 40 (11–84) 2 (0.5–9) 76 (51–83) 38 (22–65)Creatine/creatinine ratio 0.44 (0.02–1.9) 0.13 (0.02–1.2) 0.03 (0.02–0.4) 0.2 (0.02–1.2) 0.5 (0.3–0.7) 0.3 (0.05–0.4)

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

Values are expressed as median and range.a Nearly asymptomatic female patient heterozygous for OTC deWciency.

Plasma Control values n: 17 ASL, ASS, and OTC OTCa n: 1

Before n: 9 After n: 12

Arginine (�mol/L) 58 (29–133) 50 (12–140) 100 (40–184) 126GAA (�mol/L) 1.7 (0.7–2.5) 0.77 (0.03–0.87) 1.30 (1.02–2.19) 1.49Creatine (�mol/L) 76 (45–228) 59 (28–100) 44 (28–104) 77

A. Arias et al. / Molecular Genetics and Metabolism 82 (2004) 220–223 223

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

Acknowledgments

We thank C. Llordés for her invaluable help in sam-ple analyses. We also thank Dr. M. Rodés for amino acidanalyses. The Wnancial support of FIS (Grant # 2003-REDG054B-O) is acknowledged.

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