Analytical Biochemistry 331 (2004) 189–191 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.05.012 ANALYTICAL BIOCHEMISTRY Notes & Tips HPLC assay for guanidinoacetate methyltransferase Maria Grazia Alessandrì, a,¤ Lisa Celati, a Roberta Battini, a Fulvia Baldinotti, b Chike Item, c Vincenzo Leuzzi, d and Giovanni Cioni e a Dipartimento di Neuroscienze dell’Età Evolutiva, IRCCS Stella Maris, Pisa, Italy b Laboratorio di Citogenetica, Azienda Ospedaliera, Pisa, Italy c Department of Pediatrics, University Hospital and General Hospital of Vienna, Austria d Dipartimento di Scienze Neurologiche e Psichiatriche dell’Età Evolutiva, Università “La Sapienza”, Rome, Italy e Divisione di Neuropsichiatria Infantile, Università di Pisa, Pisa, Italy Received 18 December 2003 Available online 8 June 2004 Guanidinoacetate methyltransferase (GAMT; EC 2.1.1.2), 1 the enzyme catalyzing the Wnal step in creatine biosynthesis by transferring a methyl group from S- adenosylmethionine to guanidinoacetic acid (GAA), has been considered to be highly expressed in a few organs such as kidney, liver, and pancreas [1]. Low activity is reported in skeletal and cardiac muscle, mouse neuro- blastoma cells, and human fetal lung Wbroblasts [2]. The discovery of the Wrst case of GAMT deWciency [3], and more recently of other patients with the same enzymatic defect, has drawn attention to the analytical procedures used to measure the activity of this enzyme in human cells with low expression so as to avoid the more invasive hepatic biopsy. Previously published assays for GAMT activity [2,4,5] used radiolabeled molecules as substrates to reach high sensitivity and speciWcity. Moreover, all of these methods consist of numerous steps (e.g., extraction, puriWcation of the extracts, quantiWcation), require high amounts of cells, and show high unspeciWc blank radio- activity and, therefore, are more useful for detecting GAMT in tissues than in cells with low activity [2]. Ilas et al. [6] developed a speciWc and sensitive method to measure GAMT activity in human-cultured Wbroblasts, Epstein–Barr virus-transformed lymphoblasts, and culti- vated amniotic cells but still with radiolabeled substrate. In their method, creatine is separated from [ 14 C]guanidi- noacetate in HPLC and is quantiWed in a beta counter. In this article, we present a nonradioactive method for measuring GAMT activity in lymphoblasts based on the quantiWcation of creatine, the end product of the GAMT, by using HPLC with uv detector. Materials and methods Reagents. PBS-Dulbecco modiWed solution, SDS, sodium phosphate dibasic anhydrous, phosphoric acid, acetonitrile (HPLC grade), Tris–HCl, dithiothreitol (DTT), EDTA, S-adenosylmethionine, GAA, creatine, perchloric acid, bovine albumin, sodium carbonate, and sodium potassium tartrate were obtained from Sigma– Aldrich (Italy). Ultrafree Biomax-10K membranes were obtained from Millipore (Bedford, MA, USA). GH Polypro Hydrophilic Polypropylene membrane Wlters (47 mm, 0.2 m) were obtained from Gelman Sciences (Ann Arbor, MI, USA). Chromatographic conditions. A Beckman 125 dual pump equipped with a uv detector (166, Beckman) set at 210 nm, an ODS column (250 £ 4.6 mm, 5 m, Beckman), and a Rheodyne 7725i injector with a 50-l loop was used for the elution of creatine [6]. The mobile phase consisted of sodium phosphate dibasic anhydrous (7.1 g), SDS (2 g), distilled water (500ml), acetonitrile (250 ml), and phos- phoric acid to reach a Wnal pH 3; it was Wltered on GH Polypro Wlters before use. Creatine separation was per- formed isocratically at room temperature and at a Xow rate of 0.5ml/min. In these analytical conditions, the pump pressure was 0.973 K psi and the run time was 15 min. Enzyme assay. Lymphoblasts were prepared as reported in [6], with or without a concentration step in Ultrafree 4 Biomax membranes (Millipore). ¤ Corresponding author. Fax: +39-050-886247. E-mail address: mariagrazia.alessandri@inpe.unipi.it (M. Grazia Alessandrì). 1 Abbreviations used: GAMT, guanidinoacetate methyltransferase; GAA, guanidinoacetic acid; DTT, dithiothreitol.